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Sample records for lectin binding sites

  1. Histochemistry of lectin-binding sites in Halicryptus spinulosus (Priapulida).

    Science.gov (United States)

    Busch, A; Schumacher, U; Storch, V

    2001-02-01

    Priapulida represent one of the phylogenetically oldest multicellular animal groups. In multicellular animals (Metazoa) cell-to-cell and cell-to-matrix interactions are often mediated by carbohydrate residues of glycoconjugates. To analyze the carbohydrate composition of a phylogenetically old species, lectin histochemistry was employed on 5 specimens of the priapulid Halicryptus spinulosus. Many lectins bound to the chitin-containing cuticle, including those specific for carbohydrates other than N-acetylglucosamine, the principle building block of chitin. The connective tissue of the animals contained both N-acetylglucosamine and N-acetylgalactosamine. Mannose residues were widely distributed with the exception of the cuticle, but complex type carbohydrates were not present in the entire animal. Sialic acid residues were only detected in the cuticle and brush border of the intestinal epithelium, while fucose was limited to the cuticle. Thus, the lectin-binding pattern indicated that sugars typical for the linking region of both N- and O-glycoproteins in mammals are also present in H. spinulosus. Carbohydrate residues that are typical for the complex type of N-linked glycans in vertebrates are not present as are carbohydrate residues typical for the termination of O-linked carbohydrate chains. Hence, a truncated form of both N- and O-linked glycosylation is present in H. spinulosus indicating that more complex patterns of glycosylation developed later during evolution.

  2. Structure of Dioclea virgata lectin: relations between carbohydrate binding site and nitric oxide production

    International Nuclear Information System (INIS)

    Delatorre, P.; Gadelha, C.A.A.; Santi-Gadelha, T.; Nobrega, R.B.; Rocha, B.A.M.; Nascimento, K.S.; Naganao, C.S.; Sampaio, A.H.; Cavada, B.S.; Pires, A.F.; Assreuy, A.M.S.

    2012-01-01

    Full text: Lectins are proteins/glycoproteins with at least one noncatalytic domain binding reversibly to specific monosaccharides or oligosaccharides. By binding to carbohydrate moieties on the cell surface, lectins participate in a range of cellular processes without changing the properties of the carbohydrates involved. The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological proper- ties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. The DvirL diffraction analysis revealed that both the native crystal and the X-Man-complexed form are orthorhombic and belong to space group I222. The cell parameters were: a=65.4 , b=86.6 and c=90.2 (native structure), and a=61.89 , b=87.67 and c=88.78 (X-Man-complexed structure). An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration. (author)

  3. Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 onto plates coated with anti-CD4bs antibodies b12, b6 or the CD4 receptor mimetic, CD4-IgG2...

  4. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

    Energy Technology Data Exchange (ETDEWEB)

    Makyio, Hisayoshi [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan); Shimabukuro, Junpei; Suzuki, Tatsuya [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Imamura, Akihiro; Ishida, Hideharu [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Kiso, Makoto [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Ando, Hiromune, E-mail: hando@gifu-u.ac.jp [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Kato, Ryuichi, E-mail: ryuichi.kato@kek.jp [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan)

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

  5. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

    International Nuclear Information System (INIS)

    Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

    2016-01-01

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

  6. Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

    Science.gov (United States)

    Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

    2000-08-25

    Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

  7. Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules.

    Science.gov (United States)

    Nurden, A T; Horisberger, M; Savariau, E; Caen, J P

    1980-10-15

    Washed human platelets have been incubated with the lectins WGA, ConA and RCA1, adsorbed to different-sized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.

  8. Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

    DEFF Research Database (Denmark)

    Teillet, F; Lacroix, M; Thiel, Steffen

    2007-01-01

    Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To ident......Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2....... To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19...... and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3...

  9. Localization of binding sites of Ulex europaeus I, Helix pomatia and Griffonia simplicifolia I-B4 lectins and analysis of their backbone structures by several glycosidases and poly-N-acetyllactosamine-specific lectins in human breast carcinomas.

    Science.gov (United States)

    Ito, N; Imai, S; Haga, S; Nagaike, C; Morimura, Y; Hatake, K

    1996-09-01

    Several studies have shown the deletion of blood group A or B antigens and the accumulation of H antigens in human breast carcinomas. Other studies have independently demonstrated that the binding sites of lectins such as Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) are highly expressed in these cells. In order to clarify the molecular mechanisms of malignant transformation and metastasis of carcinoma cells, it is important to understand the relationship between such phenotypically distinct events. For this purpose, we examined whether the binding sites of these lectins and Ulex europaeus agglutinin I (UEA-I) are expressed concomitantly in the same carcinoma cells and analyzed their backbone structures. The expression of the binding sites of these lectins was observed independently of the blood group (ABO) of the patients and was not affected by the histological type of the carcinomas. Observation of serial sections stained with these lectins revealed that the distribution of HPA binding sites was almost identical to that of GSAI-B4 in most cases. Furthermore, in some cases, UEA-I binding patterns were similar to those of HPA and GSAI-B4 but in other cases, mosaic staining patterns with these lectins were also observed, i.e., some cell clusters were stained with both HPA and GSAI-B4 but not with UEA-I and adjacent cell clusters were stained only with UEA-I. Digestion with endo-beta-galactosidase or N-glycosidase F markedly reduced the staining intensity of these lectins. Together with the reduction of staining by these lectins, reactivity with Griffonia simplicifolia agglutinin II appeared in carcinoma cells following endo-beta-galactosidase digestion. Among the lectins specific to poly-N-acetyllactosamine, Lycopersicon esculentum agglutinin (LEA) most vividly and consistently stained the cancer cells. Next to LEA, pokeweed mitogen agglutinin was also effective in staining these cells. Carcinoma cells reactive with these

  10. Multiplicity of carbohydrate-binding sites in β-prism fold lectins ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    cancer metastasis, embryogenesis, tissue development and mitogenic stimulation ... within the sequence exhibit reasonable correlation. The distribution of the ..... II fold lectins with known structure in complex with sugar. Sugars are shown in ...

  11. Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

    Science.gov (United States)

    André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

    1999-11-01

    Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential

  12. Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

    International Nuclear Information System (INIS)

    Kohnken, R.E.; Berger, E.A.

    1987-01-01

    N-(4-Azidosalicyl) galactosamine (GalNASA), a photoactivatable, radioiodinatable analog of N-acetylgalactosamine (GalNAc), has been prepared and characterized. The authors have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a K/sub i,app/ of 800 μM, comparable to that of GalNAc. The K/sub i,app/ of GalNASA decreased to 40 μm upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl) ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125 I-GalNASA was entirely dependent upon ultraviolet light. A portion of labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethyl-enediaminetetraacetic acid. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125 I-GalNASA exhibited a K/sub d/ of 15-40 μM, in agreement with the K/sub i,app/ of prephotolyzed GalNASA observed in the carbohydrate binding assay. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc. This indicated that the location of carbohydrate-sensitive labeling within the structure of discoidin I was restricted. One particular tryptic fragment, Tr1, was examined in detail. These data suggest that Tr1 is derived from the carbohydrate binding site of discoidin I

  13. Genetics Home Reference: mannose-binding lectin deficiency

    Science.gov (United States)

    ... and Caregivers: Complement System Nobelprize.org: The Immune System - In More Detail Patient ... Sources for This Page Arora M, Munoz E, Tenner AJ. Identification of a site on mannan-binding lectin critical ...

  14. Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

    Science.gov (United States)

    Heng, M C; Fallon-Friedlander, S; Bennett, R

    1992-06-01

    Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.

  15. Distribution of binding sites for the plant lectin Ulex europaeus agglutinin I on primary sensory neurones in seven different mammalian species.

    Science.gov (United States)

    Gerke, Michelle B; Plenderleith, Mark B

    2002-01-01

    There is an increasing body of evidence to suggest that different functional classes of neurones express characteristic cell-surface carbohydrates. Previous studies have shown that the plant lectin Ulex europaeus agglutinin-I (UEA) binds to a population of small to medium diameter primary sensory neurones in rabbits and humans. This suggests that a fucose-containing glycoconjugate may be expressed by nociceptive primary sensory neurones. In order to determine the extent to which this glycoconjugate is expressed by other species, in the current study, we have examined the distribution of UEA-binding sites on primary sensory neurones in seven different mammals. Binding sites for UEA were associated with the plasma membrane and cytoplasmic granules of small to medium dorsal root ganglion cells and their axon terminals in laminae I-III of the grey matter of the spinal cord, in the rabbit, cat and marmoset monkey. However, no binding was observed in either the dorsal root ganglia or spinal cord in the mouse, rat, guinea pig or flying fox. These results indicate an inter-species variation in the expression of cell-surface glycoconjugates on mammalian primary sensory neurones.

  16. Population heterogeneity in the surface expression of Ulex europaeus I-lectin (UEA I)-binding sites in cultured malignant and transformed cells

    Energy Technology Data Exchange (ETDEWEB)

    Virtanen, I.; Lehtonen, E.; Naervaenen, O.; Leivo, I.; Lehto, V.P.

    1985-11-01

    We studied the binding of fluorochrome-coupled Ulex europaeus I-lectin (UEA-I) to cultured malignant cells: all human malignant and transformed cells and also mouse teratocarcinoma cells examined gave a homogeneous cell membrane-type of surface staining only in some of the cells. Such a population heterogeneity appeared to be independent of the cell cycle. Instead, other lectin conjugates used bound homogeneously to all cell. In permeabilized cells, a juxtanuclear reticular staining of the Golgi apparatus was seen in the UEA-I-positive cells. No staining of the pericellular matrix components, produced by malignant cells grown in serum-free culture medium, could be obtained with TRITC-UEA-I. UEA-I-lectin recognized most polypeptides from A8387 fibrosarcoma cells and HeLa cells, metabolically labelled with (/sup 3/H)fucose. Furthermore, surface labelling of these cells with the neuraminidase-galactose oxidase/sodium borohydride method disclosed that both UEA-I and Ricinus communis agglutinin I revealed the same major surface glycoproteins. Results with metabolically labelled cells showed, in addition, that UEA-I-lectin did not bind to secreted glycoproteins produced by A8387 cells and recognized by other lectins. The results indicate that transformed and malignant cells show a distinct population heterogeneity in their expression of some cell surface-associated fucosyl glycoconjugates. The results also suggest that malignant cells can glycosylate their membrane and secreted glycoproteins in a different manner.

  17. Mannose-binding lectin genetics: from A to Z

    DEFF Research Database (Denmark)

    Garred, Peter

    2008-01-01

    MBL (mannose-binding lectin) is primarily a liver-derived collagen-like serum protein. It binds sugar structures on micro-organisms and on dying host cells and is one of the four known mediators that initiate activation of the complement system via the lectin pathway. Common variant alleles situa...

  18. The distribution of lectin receptor sites in human breast lesions.

    Science.gov (United States)

    Skutelsky, E; Hoenig, S; Griffel, B; Alroy, J

    1988-08-01

    Conflicting data regarding the status of A, B, H and T antigens in epithelium of normal, mastopathies, fibroadenomas and carcinomas of the breast stimulated us to re-examine the carbohydrate residues in these condition. Currently, we extended the number of carbohydrate residues studied by using ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex (ABC) as a visualant. In addition, the pattern of lectin staining of cancerous cells in primary and metastatic sites was compared. In primary and metastatic breast carcinomas, lectin receptor sites were stained more intensely with Concanavalia ensiformi agglutinin (*Con A), Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA), than in normal breast, in mastopathies or in fibroadenomas. Cryptic receptor sites for peanut agglutinin (PNA) were stained in all cases of breast carcinomas, while free PNA sites stained only in a few cases of well-differentiated carcinomas. Receptors sites for Ulex europaeus agglutinin-I (UEA-I) stained non-malignant epithelium of patients with blood group H but did not stain malignant cells. The results show significant differences in lectin-binding patterns and staining intensities between normal and non-malignant, and malignant epithelial breast cells. Furthermore, these results indicate that in malignant cells, there is an increased content of sialic acid-rich carbohydrates but not of asialylated glycoconjugates.

  19. Sugared biomaterial binding lectins: achievements and perspectives

    Czech Academy of Sciences Publication Activity Database

    Bojarová, Pavla; Křen, Vladimír

    2016-01-01

    Roč. 4, č. 8 (2016), s. 1142-1160 ISSN 2047-4830 R&D Projects: GA ČR GC15-02578J Institutional support: RVO:61388971 Keywords : C-TYPE LECTINS * AMPHIPHILIC JANUS GLYCODENDRIMERS * BIOMEDICALLY RELEVANT LECTINS Subject RIV: CE - Biochemistry Impact factor: 4.210, year: 2016

  20. Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

    Science.gov (United States)

    Qaddoumi, Mohamed; Lee, Vincent H L

    2004-07-01

    To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

  1. Mannan-Binding Lectin in Cardiovascular Disease

    Directory of Open Access Journals (Sweden)

    Izabela Pągowska-Klimek

    2014-01-01

    Full Text Available Cardiovascular disease remains the leading cause of mortality and morbidity worldwide so research continues into underlying mechanisms. Since innate immunity and its potent component mannan-binding lectin have been proven to play an important role in the inflammatory response during infection and ischaemia-reperfusion injury, attention has been paid to its role in the development of cardiovascular complications as well. This review provides a general outline of the structure and genetic polymorphism of MBL and its role in inflammation/tissue injury with emphasis on associations with cardiovascular disease. MBL appears to be involved in the pathogenesis of atherosclerosis and, in consequence, coronary artery disease and also inflammation and tissue injury after myocardial infarction and heart transplantation. The relationship between MBL and disease is rather complex and depends on different genetic and environmental factors. That could be why the data obtained from animal and clinical studies are sometimes contradictory proving not for the first time that innate immunity is a “double-edge sword,” sometimes beneficial and, at other times disastrous for the host.

  2. Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity

    DEFF Research Database (Denmark)

    Pedersen, Johannes W; Bennett, Eric P; Schjoldager, Katrine T-B G

    2011-01-01

    UDP-GalNAc:polypeptide a-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection...... of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence...... on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate...

  3. Lectin binding patterns and immunohistochemical antigen detection ...

    African Journals Online (AJOL)

    Ibrahim Eldaghayes

    2018-02-09

    Feb 9, 2018 ... placenta and lungs of Brucella abortus-bovine infected fetuses. María Andrea ... The present lectin histochemical study revealed a distinctive pattern of oligosaccharide .... tissue was used as a positive control and nonimmune.

  4. Cholesterol Crystals Activate the Lectin Complement Pathway via Ficolin-2 and Mannose-Binding Lectin

    DEFF Research Database (Denmark)

    Pilely, Katrine; Rosbjerg, Anne; Genster, Ninette

    2016-01-01

    Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind...... CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using...... recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis....

  5. Mannan-binding lectin in astma and allergy

    DEFF Research Database (Denmark)

    Kaur, S.; Thiel, Steffen; Sarma, P.U.

    2006-01-01

    Mannan-binding lectin (MBL) is a vital and versatile component of innate immunity. It is present in serum and may bind to a plethora of microbial pathogens and mediate opsonization of these by complement-dependent and/or independent mechanisms. Low-MBL levels in serum, attributed to certain genet...

  6. Clinical manifestations of mannan-binding lectin deficiency

    DEFF Research Database (Denmark)

    Thiel, S; Frederiksen, P D; Jensenius, Jens Christian

    2006-01-01

    Mannan-binding lectin (MBL) is a plasma protein of the innate immune system with the ability to initiate antimicrobial and inflammatory actions. MBL deficiency is common. More than 10% of the general population may, depending on definition, be classified as MBL deficient, underlining the redundan...

  7. Mannan-binding lectin activates C3 and the

    DEFF Research Database (Denmark)

    Selander, B.; Martensson, U.; Weintraub, A.

    2006-01-01

    Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera...... and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S...

  8. Presenting Precision Glycomacromolecules on Gold Nanoparticles for Increased Lectin Binding

    Directory of Open Access Journals (Sweden)

    Sophia Boden

    2017-12-01

    Full Text Available Glyco-functionalized gold nanoparticles have great potential as biosensors and as inhibitors due to their increased binding to carbohydrate-recognizing receptors such as the lectins. Here we apply previously developed solid phase polymer synthesis to obtain a series of precision glycomacromolecules that allows for straightforward variation of their chemical structure as well as functionalization of gold nanoparticles by ligand exchange. A novel building block is introduced allowing for the change of spacer building blocks within the macromolecular scaffold going from an ethylene glycol unit to an aliphatic spacer. Furthermore, the valency and overall length of the glycomacromolecule is varied. All glyco-functionalized gold nanoparticles show high degree of functionalization along with high stability in buffer solution. Therefore, a series of measurements applying UV-Vis spectroscopy, dynamic light scattering (DLS and surface plasmon resonance (SPR were performed studying the aggregation behavior of the glyco-functionalized gold nanoparticles in presence of model lectin Concanavalin A. While the multivalent presentation of glycomacromolecules on gold nanoparticles (AuNPs showed a strong increase in binding compared to the free ligands, we also observed an influence of the chemical structure of the ligand such as its valency or hydrophobicity on the resulting lectin interactions. The straightforward variation of the chemical structure of the precision glycomacromolecule thus gives access to tailor-made glyco-gold nanoparticles (glyco-AuNPs and fine-tuning of their lectin binding properties.

  9. Mannan-binding lectin and mannan-binding lectin-associated serine protease 2 in acute pancreatitis

    DEFF Research Database (Denmark)

    Novovic, Srdan; Andersen, Anders; Ersbøll, Annette Kjær

    2011-01-01

    Complement activation may play a prominent role in acute pancreatitis (AP). Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) participate in complement activation. The objective of the present study was to evaluate the role of MBL and MASP-2 as markers in AP with regard...

  10. Chitovibrin: a chitin-binding lectin from Vibrio parahemolyticus.

    Science.gov (United States)

    Gildemeister, O S; Zhu, B C; Laine, R A

    1994-12-01

    A novel 134 kDa, calcium-independent chitin-binding lectin, 'chitovibrin', is secreted by the marine bacterium Vibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers > dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0-4 M NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bind V. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.

  11. Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

    Science.gov (United States)

    Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

    2017-01-01

    Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

  12. Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

    Directory of Open Access Journals (Sweden)

    Yoko Itakura

    2017-05-01

    Full Text Available Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine. Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

  13. Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

    International Nuclear Information System (INIS)

    Mikeska, Ruth; Wacker, Roland; Arni, Raghuvir; Singh, Tej P.; Mikhailov, Albert; Gabdoulkhakov, Azat; Voelter, Wolfgang; Betzel, Christian

    2004-01-01

    The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R free = 23.6%) and 20.9 (R free = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound

  14. Mannan-binding lectin and healing of a radiation-induced chronic ulcer--a case report on mannan-binding lectin replacement therapy

    DEFF Research Database (Denmark)

    Maaløe, Nanna; Bonde, C; Laursen, I

    2011-01-01

    Mannan-binding lectin is an important component of innate immunity, and insufficiency is associated with several clinical disorders. Recently, experimental replacement therapy with plasma-derived mannan-binding lectin has become an option. The current article presents the case of a patient with a...

  15. A Computational Study of the Oligosaccharide Binding Sites in the Lectin-Like Domain of Tumor Necrosis Factor and the TNF-derived TIP Peptide

    Czech Academy of Sciences Publication Activity Database

    Dulebo, A.; Ettrich, Rüdiger; Lucas, R.; Kaftan, D.

    2012-01-01

    Roč. 18, č. 27 (2012), s. 4236-4243 ISSN 1381-6128 Institutional support: RVO:67179843 Keywords : lectin-like domain * tumor necrosis factor * TIP peptide * oligosaccharides * molecular docking * molecular dynamics simulation * alveolar liquid clearance Subject RIV: CE - Biochemistry Impact factor: 3.311, year: 2012

  16. Changes in cell surface structure by viral transformation studied by binding of lectins differing in sugar specificity.

    Science.gov (United States)

    Tsuda, M; Kurokawa, T; Takeuchi, M; Sugino, Y

    1975-10-01

    Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown in coverslips. The following 125I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetyl-galactosamine). Cells for a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure. The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

  17. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

    Science.gov (United States)

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  18. Immunogold study on lectin binding in the porcine zona pellucida and granulosa cells

    Directory of Open Access Journals (Sweden)

    F Parillo

    2009-06-01

    Full Text Available An ultrastructural localization of lectin receptors on the zona pellucida (ZP of porcine antral oocytes and on the granulosa cells was performed using a panel of horseradish peroxidase- labelled lectins in conjunction with antiperoxidase antibody and protein A-gold. In some cases, lectin incubation was preceded by sialidase digestion. WGA-, Con-A-, UEA-I-, RCA-I-, PNA- and SBA-reactive sites were distributed differently in the porcine ZP. Sialidase digestion increased the positivity obtained with RCA-I and it was necessary to promote PNA and SBA reactivity. These results indicated that the ZP contained N-acetylglucosamine, a-mannose, a- fucose, b-Gal-(1-4GlcNAc, b-Gal- (1-3GalNAc, b-GalNAc and sialic acid residues. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the ZP, thus suggesting that the oocyte and granulosa cells are the site of synthesis of ZP glucidic determinants.

  19. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  20. Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis

    DEFF Research Database (Denmark)

    Tawakoli, Pune Nina; Neu, Thomas R; Busck, Mette Marie

    2017-01-01

    lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence......The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled......-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates...

  1. Mannose-binding lectin polymorphisms and susceptibility to infection in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Garred, P; Madsen, H O; Halberg, P

    1999-01-01

    To determine whether variant alleles in the coding portion of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to systemic lupus erythematosus (SLE) and concomitant infections.......To determine whether variant alleles in the coding portion of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to systemic lupus erythematosus (SLE) and concomitant infections....

  2. Mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Øhlenschlaeger, Tommy; Garred, Peter; Madsen, Hans O

    2004-01-01

    Cardiovascular disease is an important complication in patients with systemic lupus erythematosus (SLE). Variant alleles of the mannose-binding lectin gene are associated with SLE as well as with severe atherosclerosis. We determined whether mannose-binding lectin variant alleles were associated...

  3. Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

    Science.gov (United States)

    Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

    2012-01-01

    Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

  4. Expression of binding properties of Gal/GalNAc reactive lectins by mammalian glycotopes (an updated report).

    Science.gov (United States)

    Wu, A M

    2001-01-01

    Expression of the binding properties of Gal/GalNAc specific lectins, based on the affinity of decreasing order of mammalian glycotopes (determinants) rather than monosaccharide inhibition pattern, is probably one of the best ways to express carbohydrate specifity and should facilitate the selection of lectins as structural probes for studying mammalian glycobiology. Eleven mammalian structural units have been selected to express the binding domain of applied lectins. They are: 1. F, GalNAcalpha1 --> 3GalNAc; 2. A, GalNAcalpha1 --> 3Gal; 3. T, Galbeta1 --> 3GalNAc; 4. I, Galbeta 1 --> 3GlcNAc; 5. II, Galbeta1 --> 4GlcNAc; 6. B, Galalpha1 --> 3Gal; 7. E, Galalpha1--> 4Gal; 8. L, Galbeta1 --> 4Glc; 9. P, GalNAcbeta1 --> 3Gal; 10. S, GalNAcbeta1 --> 4Gal and 11. Tn, GalNAcalpha1 --> 4Ser (Thr) of the peptide chain. Thus, the carbohydrate specificity of Gal/GalNAc reactive lectins can be divided into classes according to their highest affinity for the above disaccharides and/or Tn residue. Examples of the binding properties of these lectins can be demonstrated by Ricimus communis agglutinin (RCA1), grouped as II specific lectin and its binding property is II > I > B > T; Ahrus precatorius agglutinin (APA), classified as T and its carbohydrate specificity is T > I/II > E > B > Tn; Artocarpus integrifolia (jacalin, AIL), as T/Tn specific and its binding reactivity is T > Tn > I (II) and Geodia cydonium (GCL), as F/A specific, and with affinity for F > Ah [GalNAcalpha1-->43(L(Fuc)alpha1-->2)Gal] > I > L. Due to the multiple reactivity of lectins toward mammalian glycotopes, the possible existence of different combining sites or subsites in the same molecule has to be examined, and the differential binding properties of these combining sites (if any) have to be characterized. To establish the relationship among the amino acid sequences of the combining sites of plant lectins and mammalian glycotopes should be an important direction to be addressed in lectinology.

  5. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Morrison, A.I.; Keeble, S.; Watt, F.M.

    1988-01-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

  6. Human mannose-binding lectin inhibitor prevents Shiga toxin-induced renal injury

    DEFF Research Database (Denmark)

    Ozaki, Masayuki; Kang, Yulin; Tan, Ying Siow

    2016-01-01

    Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a cr...

  7. Plant lectins: the ties that bind in root symbiosis and plant defense.

    Science.gov (United States)

    De Hoff, Peter L; Brill, Laurence M; Hirsch, Ann M

    2009-07-01

    Lectins are a diverse group of carbohydrate-binding proteins that are found within and associated with organisms from all kingdoms of life. Several different classes of plant lectins serve a diverse array of functions. The most prominent of these include participation in plant defense against predators and pathogens and involvement in symbiotic interactions between host plants and symbiotic microbes, including mycorrhizal fungi and nitrogen-fixing rhizobia. Extensive biological, biochemical, and molecular studies have shed light on the functions of plant lectins, and a plethora of uncharacterized lectin genes are being revealed at the genomic scale, suggesting unexplored and novel diversity in plant lectin structure and function. Integration of the results from these different types of research is beginning to yield a more detailed understanding of the function of lectins in symbiosis, defense, and plant biology in general.

  8. Toxicity and Binding Profile of Lectins from the Genus Canavalia on Brine Shrimp

    Directory of Open Access Journals (Sweden)

    Francisco Vassiliepe Sousa Arruda

    2013-01-01

    Full Text Available Lectins are sugar-binding proteins widely distributed in nature with many biological functions. Although many lectins have a remarkable biotechnological potential, some of them can be cytotoxic. Thus, the aim of this study was to assess the toxicity of five lectins, purified from seeds of different species of Canavalia genus. In order to determine the toxicity, assays with Artemia nauplii were performed. In addition, a fluorescence assay was carried out to evaluate the binding of lectins to Artemia nauplii. In order to verify the relationship between the structure of lectins and their cytotoxic effect, structural analysis was carried out to evaluate the volume of the carbohydrate recognition domain (CRD of each lectin. The results showed that all lectins exhibited different toxicities and bound to a similar area in the digestive tract of Artemia nauplii. Concerning the structural analysis, differences in spatial arrangement and volume of CRD may explain the variation of the toxicity exhibited by each lectin. To this date, this is the first study that establishes a link between toxicity and structure of CRD from Diocleinae lectins.

  9. [Lectin-binding patterns and cell kinetics of head and neck squamous cell carcinomas].

    Science.gov (United States)

    Gotoh, T

    1991-01-01

    In order to elucidate the cell characteristics of head and neck squamous cell carcinomas, the cell kinetics and lectin binding patterns were compared with the histological classification and staging of the tumors, using surgically resected materials (maxillary sinus 10, oral cavity 21, pharynx 8, larynx 11). Eight biotinylated lectins (WGA, 1-PHA, ConA, UEA1, RCA1, SBA, DBA, PNA) were applied to the paraffin-embedded sections, and were visualized histochemically by the streptavidin-alkaline phosphatase method. The DNA contents of the isolated carcinoma cells obtained from the adjacent thick sections were evaluated using an epi-illumination cytofluorometer after propidium iodide staining. On lectin histochemistry, the binding pattern of WGA lectin was similar between carcinoma tissues and normal tissues, but the binding was more intense in well differentiated than less differentiated carcinomas. Lymph node metastasis was found to be related to the presence of cells with poor WGA-binding. In the binding patterns of the other lectins, RCA1, SBA and ConA were related to the differentiation of carcinomas, but they were not related to the TNM-classification. DNA cytofluorometry exhibited marked polyploidization, which progressed with the advancement of the clinical and pathological staging of carcinomas. However, the DNA ploidy pattern was not associated with the cell characteristics such as the degree of histological differentiation and the lectin-binding pattern, except that the appearance of aneuploidy had some relationship with the binding-patterns of UEA1 and 1-PHA.

  10. Crosstalk between innate and adaptive immune responses to infectious bronchitis virus after vaccination and challenge of chickens varying in serum mannose-binding lectin concentrations

    DEFF Research Database (Denmark)

    Juul-Madsen, Helle R.; Norup, Liselotte R.; Jørgensen, Poul Henrik

    2011-01-01

    Mannose-binding lectin (MBL), a C-type collectin with structural similarities to C1q, is an innate pattern-recognition molecule that is sequestered to sites of inflammation and infections. MBL selectively binds distinct chemical patterns, including carbohydrates expressed on all kinds of pathogen...

  11. Binding of navy bean (Phaseolus vulgaris) lectin to the intestinal cells of the rat and its effect on the absorption of glucose

    International Nuclear Information System (INIS)

    Donatucci, D.A.; Liener, I.E.; Gross, C.J.

    1987-01-01

    The main objectives of this investigation were to study the binding of a lectin from navy beans with the epithelial cells of the rat intestine and to assess the effect of such binding on the ability of the intestine to absorb glucose. A Scatchard plot, based on the binding of 125 I-labeled lectin to isolated intestinal epithelial cells, was used to calculate an association constant (Ka) of 15 x 10(6)M-1 and the number of binding sites per cell, 12 x 10(6). Metabolic studies were conducted over a period of 5 d on groups of rats fed raw or autoclaved navy bean flour and casein with or without the purified lectin. Growth, protein digestibility, biological value and net protein utilization were significantly lower in animals that had been fed raw navy bean flour or casein plus lectin than in control groups fed diets containing autoclaved navy bean flour or casein alone. Vascular perfusion was used to measure the rate of uptake of glucose by the intestines of rats that had received the various dietary treatments. The rate of absorption of [ 14 C]glucose by intestines from rats fed raw navy bean flour or casein plus lectin was approximately one-half that of their counterparts fed the autoclaved flour or casein alone. These results provide evidence that the lectin, by virtue of its interference with intestinal absorption, is responsible, at least in part, for the nutritional inferiority of raw navy beans

  12. New perspectives on mannan-binding lectin-mediated complement activation

    DEFF Research Database (Denmark)

    Degn, Søren Egedal; Thiel, Steffen; Jensenius, Jens Christian

    2007-01-01

    The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP......, allowing C3 activation in the absence of components otherwise believed critical. The classical bypass pathways are dependent on C1 and components of the AP. A recent study has shown the existence also of a lectin bypass pathway dependent on mannan-binding lectin (MBL) and AP components. The emerging...

  13. Low mannose-binding lectin serum levels are associated with reduced kidney graft survival

    DEFF Research Database (Denmark)

    Bay, Jakob Thaning; Sørensen, Søren S; Hansen, Jesper M

    2013-01-01

    Activation of the complement system is initiated by the alternative, the classical, or the lectin pathway. As the complement system is involved in the pathophysiology of graft rejection after kidney transplantation, we investigated the possible role of mannose-binding lectin in kidney transplanta...... immunity in maintaining kidney graft survival, but these are probably overruled by HLA immunization.Kidney International advance online publication, 21 November 2012; doi:10.1038/ki.2012.373....

  14. Oxidative stress decreases functional airway mannose binding lectin in COPD.

    Directory of Open Access Journals (Sweden)

    Hai B Tran

    Full Text Available We have previously established that a defect in the ability of alveolar macrophages (AM to phagocytose apoptotic cells (efferocytosis and pathogens is a potential therapeutic target in COPD. We further showed that levels of mannose binding lectin (MBL; required for effective macrophage phagocytic function were reduced in the airways but not circulation of COPD patients. We hypothesized that increased oxidative stress in the airway could be a cause for such disturbances. We therefore studied the effects of oxidation on the structure of the MBL molecule and its functional interactions with macrophages. Oligomeric structure of plasma derived MBL (pdMBL before and after oxidation (oxMBL with 2,2'-azobis(2-methylpropionamidinedihydrochroride (AAPH was investigated by blue native PAGE. Macrophage function in the presence of pd/oxMBL was assessed by measuring efferocytosis, phagocytosis of non-typeable Haemophilus influenzae (NTHi and expression of macrophage scavenger receptors. Oxidation disrupted higher order MBL oligomers. This was associated with changed macrophage function evident by a significantly reduced capacity to phagocytose apoptotic cells and NTHi in the presence of oxMBL vs pdMBL (eg, NTHi by 55.9 and 27.0% respectively. Interestingly, oxidation of MBL significantly reduced macrophage phagocytic ability to below control levels. Flow cytometry and immunofluorescence revealed a significant increase in expression of macrophage scavenger receptor (SRA1 in the presence of pdMBL that was abrogated in the presence of oxMBL. We show the pulmonary macrophage dysfunction in COPD may at least partially result from an oxidative stress-induced effect on MBL, and identify a further potential therapeutic strategy for this debilitating disease.

  15. Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom.

    Science.gov (United States)

    Sartim, Marco A; Pinheiro, Matheus P; de Pádua, Ricardo A P; Sampaio, Suely V; Nonato, M Cristina

    2017-02-01

    BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86-Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Science.gov (United States)

    Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

    2005-01-01

    Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent

  17. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Directory of Open Access Journals (Sweden)

    Shin Soojung

    2005-07-01

    Full Text Available Abstract Background Pluripotent human embryonic stem cells (hESCs have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4, to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomatoesculetum lectin (TL, Ricinus communis agglutinin (RCA, and Concanavalin A (Con A bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA and Lotus tetragonolobus lectin (LTL did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L, Vicia villosa agglutinin (VVA, Ulex europaeus agglutinin (UEA, Phaseolus vulgaris erythro-agglutinin (PHA-E, and Maackia amurensis agglutinin (MAA bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the

  18. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    Science.gov (United States)

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

    2004-01-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  19. Mannan-binding lectin in cerebrospinal fluid: a leptomeningeal protein

    Directory of Open Access Journals (Sweden)

    Reiber Hansotto

    2012-08-01

    Full Text Available Abstract Background Mannan-binding lectin (MBL, a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb, i.e., with CSF flow rate. Methods MBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction. Results MBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3 The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66% compared to the MBL concentrations in serum (CV = 146% indicate an independent source of MBL in CSF. 4 The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb. Conclusions MBL in CSF is

  20. Tetranectin, a trimeric plasminogen-binding C-type lectin

    DEFF Research Database (Denmark)

    Holtet, T L; Graversen, Jonas Heilskov; Clemmensen, I

    1997-01-01

    -linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization....

  1. Lectin binders

    International Nuclear Information System (INIS)

    Rudiger, H.; Gebauer, G.; Gansera, R.; Schurz, H.; Schimpl, A.

    1982-01-01

    Lectins are widely distributed in the plant kingdom, many of them being well characterized in their chemical structure and the effects they have on alien biological systems such as erythrocytes or lymphocytes. The biological function of plant lectins remains speculative. We therefore inspected plant extracts from components which might bind specifically to the lectin from the respective plant. Single proteins (lectin binders) could be isolated from each plant extract. The interaction of these proteins with lectins was demonstrated and qualified by several methods. Similar to the lectins, the lectin binders are localized in the cytoplasm in contrast to them, however, they persist during germination and plant growth. Their precise role in the plant is not known, but they are likely to be associated with lectins not only in vitro but also in vivo. They also interact with alien cells, and are able to stimulate mitosis in murine lymphocytes. Some lectin binders act specifically on B lymphocytes, leaving T cells uninfluenced

  2. A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

    Science.gov (United States)

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1995-04-01

    The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

  3. Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus

    DEFF Research Database (Denmark)

    Juul-Madsen, H.R.; Munch, M.; Handberg, Kurt

    2003-01-01

    Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonizati...

  4. Mannose-binding lectin impairs Leptospira activity through the inhibitory effect on the motility of cell.

    Science.gov (United States)

    Xu, Jun; Guo, Yijie; Nakamura, Shuichi; Islam, Md Shafiqul; Tomioka, Rintaro; Yoneyama, Hiroshi; Isogai, Emiko

    2015-02-01

    Mannose-binding lectin (MBL) plays key role in lectin pathway of innate immunity, and shows the ability of triggering opsonization intermediately. Substantial increase in the serum level of MBL has been confirmed during leptospirosis, which caused by a pathogenic spirochete, Leptospira. Leptospira has a fascinating locomotion pattern, which simultaneously gyrating and swimming forward, such motility enables that Leptospira is difficult to be captured by immune cells if without any assistance. In this study, the effect of mannose-binding lectin to Leptospira was quantitatively investigated by measuring some kinematic parameters, to discover the mechanism behind MBL-mediated immune responses during leptospiral infection. The results showed that mannose-binding lectin is capable of inhibiting the motility of Leptospira by transforming free swimming cells to tumbled rotating cells, resulted in the increase number of rotating cells. Otherwise, decrease in rotation rate of rotating cell has been observed. However, the swimming speed of swimming Leptospira cells showed no observable change under the effect of MBL. The inhibitory effect were only valid in a relatively short period, Leptospira cells regained their original motility after 2 h. This raises an interesting topic that Leptospira is somehow able to escape from the inhibitory effect of MBL by dragging such unfavorable molecules toward to the cell end and eventually throwing it out. The inhibitory effect of MBL on the motility of Leptospira is expected to provide a new insight into lectin pathway. Copyright © 2015 Elsevier GmbH. All rights reserved.

  5. Differing lectin-binding patterns of malignant melanoma and nevocellular and Spitz nevi.

    Science.gov (United States)

    Kohchiyama, A; Oka, D; Ueki, H

    1987-01-01

    The lectin-binding patterns of primary malignant melanoma, nevocellular nevus, and Spitz nevus were studied on formalin-fixed, paraffin-embedded sections using a series of biotinylated lectins--concanavalin A (ConA), Ricinus communis agglutinin-1 (RCA1), dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), maclura pomifera agglutinin (MPA), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and Ulex europeus agglutinin-1(UEA1)--and employing the avidin-biotin-peroxidase complex method. In nevocellular and Spitz nevi, all of the nevus cells were positively stained with ConA and RCA1. No positive staining was observed, however, with the other lectins and no change in binding patterns occurred following neuraminidase pretreatment. In malignant melanoma, all of the melanoma cells were positively stained with ConA and RCA1, and some were also stained with MPA, PNA, and WGA. In addition, DBA, SBA, MPA, PNA, and WGA labeled all of the melanoma cells after neuraminidase pretreatment. No positive staining was observed with UEA1 despite neuraminidase pretreatment. The present results showed that malignant melanoma and nevocellular and Spitz nevi have different lectin-binding patterns and different responses to neuraminidase pretreatment. We, therefore, believe that the lectin staining on paraffin-embedded sections can be a useful probe for the differentiation of these diseases.

  6. Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

    Science.gov (United States)

    Wu, A M; Song, S C; Sugii, S; Herp, A

    1997-01-01

    Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E

  7. Effects of mannose-binding lectin polymorphisms on irinotecan-induced febrile neutropenia

    NARCIS (Netherlands)

    J.M. van der Bol (Jessica); M.J.A. de Jonge (Maja); R.H.N. van Schaik (Ron); A. Sparreboom (Alex); M.A. van Fessem (Marianne); F.E. Geijn (Fleur); P.L.A. van Daele (Paul); J. Verweij (Jaap); S. Sleijfer (Stefan); A.H.J. Mathijssen (Ron)

    2010-01-01

    textabstractObjective. Mannose-binding lectin (MBL) is important in the innate immune response. MBL2 gene polymorphisms affect MBL expression, and genotypes yielding low MBL levels have been associated with an elevated risk for infections in hematological cancer patients undergoing chemotherapy.

  8. Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

    DEFF Research Database (Denmark)

    Kirkeby, Svend; André, Sabine; Gabius, Hans-Joachim

    2004-01-01

    OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue. DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including...

  9. Lectin-binding characteristics of a Lyme borreliosis spirochete Borrelia burgdorferi sensu stricto

    Czech Academy of Sciences Publication Activity Database

    Vancová, M.; Nebesářová, J.; Grubhoffer, Libor

    2005-01-01

    Roč. 50, č. 3 (2005), s. 229-238 ISSN 0015-5632 R&D Projects: GA ČR GA206/03/1323; GA AV ČR KSK6005114 Institutional research plan: CEZ:AV0Z60220518 Keywords : Borrelia burgdorferi * electron microscopy * lectin binding Subject RIV: EE - Microbiology, Virology Impact factor: 0.918, year: 2005

  10. Mannose-binding lectin and infection risk in newborns: a systematic review

    NARCIS (Netherlands)

    Israëls, J.; Frakking, F. N. J.; Kremer, L. C. M.; Offringa, M.; Kuijpers, T. W.; van de Wetering, M. D.

    2010-01-01

    The authors systematically reviewed the literature on mannose-binding lectin (MBL) and infections in newborns to determine whether infection risk is increased in MBL-deficient newborns. All original reports on MBL and infections in newborns were retrieved from Embase, Medline and CENTRAL from 1966

  11. Low serum mannose-binding lectin level increases the risk of death due to pneumococcal infection

    DEFF Research Database (Denmark)

    Eisen, Damon P; Dean, Melinda M; Boermeester, Marja A

    2008-01-01

    BACKGROUND: Previous studies have shown associations between low mannose-binding lectin (MBL) level or variant MBL2 genotype and sepsis susceptibility. However, MBL deficiency has not been rigorously defined, and associations with sepsis outcomes have not been subjected to multivariable analysis....

  12. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Unknown

    of kit protocol except that the RT step was prolonged for a further reaction ... ing a dA-tailing Kit (Sangon). DNA ligation with ... RT-PCR amplification was performed three times. 2.8 Expression of ..... of a new mannose-binding lectin gene from Taxus media; J. Biosci. ... ing: A Laboratory Manual, 2nd edition (New York: Cold.

  13. Preoperative mannan-binding lectin pathway and prognosis in colorectal cancer

    DEFF Research Database (Denmark)

    Ytting, Henriette; Christensen, Ib Jarle; Jensenius, Jens Christian

    2005-01-01

    PURPOSE: Deficiency of the mannan-binding lectin (MBL) pathway of innate immunity is associated with increased susceptibility to infections. In patients with colorectal cancer (CRC), postoperative infection is associated with poor prognosis. The aim of the present study was to evaluate (1...

  14. Mannan-binding lectin polymorphisms and serum levels in patients with endometriosis

    DEFF Research Database (Denmark)

    Kruse, Christina; Steffensen, Rudi; Nielsen, Hans J

    2014-01-01

    OBJECTIVE: To investigate a possible association between endometriosis and low levels of mannan-binding lectin (MBL). STUDY DESIGN: Case-control study of blood samples from 100 patients with endometriosis compared with results from a group of 350 blood donors. RESULT: The frequency of MBL levels...... endometriosis and low levels of MBL....

  15. [Inhibition by cysteine of the carbohydrate-binding activity of lectins from Ricinus communis, Canavalia ensiformis and Euonymus europaeus].

    Science.gov (United States)

    Dvorkin, V M

    1985-10-01

    Precipitation induced by different lectins has been studied in the presence of some aminoacids. It was shown that precipitates formed by lectins from Ricinus communis (RCA1), Canavalia ensiformis (Con A), Euonymus europaeus (Eel) in the presence of appropriate carbohydrate-containing molecules disappeared after cysteine addition, like after addition of specific carbohydrate precipitation inhibitors. It is assumed that cysteine residues of RCA1, Con A and Eel lectins are essential for their carbohydrate binding activity.

  16. Mannose-binding lectin deficiency and acute exacerbations of chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Woodruff PG

    2012-11-01

    Full Text Available Richard K Albert,1 John Connett,2 Jeffrey L Curtis,3,4 Fernando J Martinez,3 MeiLan K Han,3 Stephen C Lazarus,5 Prescott G Woodruff51Medicine Service, Denver Health and Department of Medicine, University of Colorado Denver, Denver, CO, 2Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN, 3Pulmonary and Critical Care Medicine, Department of Medicine, University of Michigan, Ann Arbor, MI, 4Pulmonary and Critical Care Medicine, VA Medical Center, Ann Arbor, MI, 5Pulmonary and Critical Care Medicine, Department of Medicine, and Cardiovascular Research Institute, University of California, San Francisco, CA, USABackground: Mannose-binding lectin is a collectin involved in host defense against infection. Whether mannose-binding lectin deficiency is associated with acute exacerbations of chronic obstructive pulmonary disease is debated.Methods: Participants in a study designed to determine if azithromycin taken daily for one year decreased acute exacerbations had serum mannose-binding lectin concentrations measured at the time of enrollment.Results: Samples were obtained from 1037 subjects (91% in the trial. The prevalence of mannose-binding lectin deficiency ranged from 0.5% to 52.2%, depending on how deficiency was defined. No differences in the prevalence of deficiency were observed with respect to any demographic variable assessed, and no differences were observed in time to first exacerbation, rate of exacerbations, or percentage of subjects requiring hospitalization for exacerbations in those with deficiency versus those without, regardless of how deficiency was defined.Conclusion: In a large sample of subjects with chronic obstructive pulmonary disease selected for having an increased risk of experiencing an acute exacerbation of chronic obstructive pulmonary disease, only 1.9% had mannose-binding lectin concentrations below the normal range and we found no association between mannose-binding lectin

  17. Mutational analysis of the pumpkin (Cucurbita maxima) phloem exudate lectin, PP2 reveals Ser-104 is crucial for carbohydrate binding.

    Science.gov (United States)

    Bobbili, Kishore Babu; Bandari, Shyam; Grobe, Kay; Swamy, Musti J

    2014-07-18

    The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Galactose-binding lectin from mulberry (Morus alba L. seeds with growth hormone-like activity

    Directory of Open Access Journals (Sweden)

    E. Khurtsidze

    2017-03-01

    Full Text Available Plant lectins are well documented to participate in multiple physiological activities based on selective binding to the carbohydrate structures. They have been reported to play significant roles in various processes such as growth and development, differentiation and plant protection. Nevertheless, the intrinsic roles of plant lectins still remain undefined. We purified a galactose-binding lectin, named MAL, from mulberry (M. alba L. seeds and analyzed its properties. The lectin is composed of one polypeptide of 17 kDa, which is abundant in the seed protein fraction. MAL interacted with GalNAc and galactose residues of saccharides with high binding ability. Western blotting analysis suggested that MAL is deposited in the mulberry leaves and inflorescence. MAL was examined for growth stimulatory activity on mulberry hypocotyls and internodal sections of in vitro grown P. euphratica cultures. Elongation of mulberry hypocotyls was detected in the apical parts of the hypocotyl, where the growth increment was 58%. MAL had no significant effect on the stem elongation and induction of new leaves. Our results suggest that MAL may be involved in the growth and cell elongation at initial stages of tissue development.

  19. The typing of Staphylococcus epidermidis by a lectin-binding assay

    DEFF Research Database (Denmark)

    Jarløv, J O; Hansen, J E; Rosdahl, V T

    1992-01-01

    A new typing method for Staphylococcus epidermidis was developed. Four biotinylated lectins--wheat germ agglutinin (WGA), soy bean agglutinin (SBA), lentil agglutinin (LCA) and Concanavalin A (ConA)--were added to immobilised whole cells of coagulase-negative staphylococci (CNS) in microtitration...... plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71...... strains of CNS, including 64 strains of S. epidermidis, were detected if all typing methods were taken into consideration. If only one typing method was used the highest discriminatory power among the S. epidermidis isolates was obtained with the lectin-binding assay which allowed 49 different strains...

  20. An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2

    DEFF Research Database (Denmark)

    Zlocowski, Natacha; Lorenz, Virginia; Bennett, Eric Paul

    2013-01-01

    Abstract Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif which is present in R-type lectin group. Acetylation site K521 is part of the QKW motif of ß......-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and aGalNAc-glycosylated mucin peptides...... indicated that degree of interaction of lectin domain with aGalNAc depends on the peptide sequence of mucin. Studies of inhibitory effect of various carbohydrates on interactions of ppGalNAc-T2 with MUC1aGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for aGalNAc...

  1. Mannan-binding lectin is involved in the protection against renal ischemia/ reperfusion injury by dietary restriction

    NARCIS (Netherlands)

    Shushimita; P. van der Pol (Pieter); R.W.F. de Bruin (Ron); J.N.M. IJzermans (Jan); C. van Kooten (Cees); F.J.M.F. Dor (Frank)

    2015-01-01

    textabstractPreoperative fasting and dietary restriction offer robust protection against renal ischemia/ reperfusion injury (I/RI) in mice.We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on

  2. Mannose-binding lectin (MBL2) and ficolin-2 (FCN2) polymorphisms in patients on peritoneal dialysis with staphylococcal peritonitis

    NARCIS (Netherlands)

    Meijvis, Sabine C. A.; Herpers, Bjorn L.; Endeman, Henrik; de Jong, Ben; van Hannen, Erik; van Velzen-Blad, Heleen; Krediet, Raymond T.; Struijk, Dirk G.; Biesma, Douwe H.; Bos, Willem Jan W.

    Background. Mannose-binding lectin (MBL) and ficolin-2 (FCN) are activators of the lectin pathway of complement and act as primary defences against infection. Single-nucleotide polymorphisms (SNPs) in the MBL2 and FCN2 genes influence the functionality of the proteins. Both proteins are capable of

  3. Impact of Mannose-Binding Lectin Deficiency on Radiocontrast-Induced Renal Dysfunction

    Directory of Open Access Journals (Sweden)

    Michael Osthoff

    2013-01-01

    Full Text Available Contrast-induced nephropathy (CIN is the third leading cause of acute renal failure in hospitalized patients. Endothelial dysfunction, renal medullary ischemia, and tubular toxicity are regarded as the most important factors in the pathogenesis of CIN. Mannose-binding lectin (MBL, a pattern recognition protein of the lectin pathway of complement, has been found to aggravate and mediate tissue damage during experimental renal ischemia/reperfusion (I/R injury which was alleviated by inhibition with C1 inhibitor, a potent MBL, and lectin pathway inhibitor. In this paper, we highlight the potential role of MBL in the pathogenesis of human CIN. In experimental I/R models, MBL was previously found to induce tubular cell death independent of the complement system. In addition, after binding to vascular endothelial cells, MBL and its associated serine proteases were able to trigger a proinflammatory reaction and contribute to endothelial dysfunction. In humans, urinary MBL was increased after administration of contrast media and in individuals with CIN. Moreover, individuals with normal/high MBL levels were at increased risk to develop radiocontrast-induced renal dysfunction. Hence, MBL and the lectin pathway seem to be a promising target given that a licensed, powerful, human recombinant inhibitor exits to be added to the scarce armamentarium currently available for prophylaxis of CIN.

  4. Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio (Devario aequipinnatus) and Zebrafish (Danio rerio) Hearts

    Science.gov (United States)

    Manalo, Trina; May, Adam; Quinn, Joshua; Lafontant, Dominique S.; Shifatu, Olubusola; He, Wei; Gonzalez-Rosa, Juan M.; Burns, Geoffrey C.; Burns, Caroline E.; Burns, Alan R.; Lafontant, Pascal J.

    2016-01-01

    Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins—wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)—in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins’ ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. PMID:27680670

  5. Isothermal titration calorimetric and computational studies on the binding of chitooligosaccharides to pumpkin (Cucurbita maxima) phloem exudate lectin.

    Science.gov (United States)

    Narahari, Akkaladevi; Singla, Hitesh; Nareddy, Pavan Kumar; Bulusu, Gopalakrishnan; Surolia, Avadhesha; Swamy, Musti J

    2011-04-14

    The interaction of chitooligosaccharides [(GlcNAc)(2-6)] with pumpkin phloem exudate lectin (PPL) was investigated by isothermal titration calorimetry and computational methods. The dimeric PPL binds to (GlcNAc)(3-5) with binding constants of 1.26-1.53 × 10(5) M(-1) at 25 °C, whereas chitobiose exhibits approximately 66-fold lower affinity. Interestingly, chitohexaose shows nearly 40-fold higher affinity than chitopentaose with a binding constant of 6.16 × 10(6) M(-1). The binding stoichiometry decreases with an increase in the oligosaccharide size from 2.26 for chitobiose to 1.08 for chitohexaose. The binding reaction was essentially enthalpy driven with negative entropic contribution, suggesting that hydrogen bonds and van der Waals' interactions are the main factors that stabilize PPL-saccharide association. The three-dimensional structure of PPL was predicted by homology modeling, and binding of chitooligosaccharides was investigated by molecular docking and molecular dynamics simulations, which showed that the protein binding pocket can accommodate up to three GlcNAc residues, whereas additional residues in chitotetraose and chitopentaose did not exhibit any interactions with the binding pocket. Docking studies with chitohexaose indicated that the two triose units of the molecule could interact with different protein binding sites, suggesting formation of higher order complexes by the higher oligomers of GlcNAc by their simultaneous interaction with two protein molecules.

  6. Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin.

    Science.gov (United States)

    Townsend, R; Read, R C; Turner, M W; Klein, N J; Jack, D L

    2001-05-01

    Deficiency of the innate, humoral immune component mannose-binding lectin (MBL) predisposes individuals to a variety of infections, but the importance of MBL in infection by anaerobes has not been addressed. The attachment of MBL to a wide range of anaerobic bacteria associated with human disease and colonization was surveyed. The results suggest that for the species we examined, resistance to MBL binding may be associated with organisms that are more commonly pathogenic and that MBL binding to some bacteria may be phase variable.

  7. Identification of structural and secretory lectin-binding glycoproteins of normal and cancerous human prostate.

    Science.gov (United States)

    Lad, P M; Cooper, J F; Learn, D B; Olson, C V

    1984-12-07

    We have utilized the technique of lectin-loading of SDS gels with iodinated concanavalin A and wheat germ agglutinin to identify glycoproteins in prostatic and seminal fluids as well as in prostate tissue fractions. The following subunits which bound both lectins were detected: (a) 50, 43 and 38 kDa subunits common to prostatic and seminal fluids, and an additional 55 kDa subunit which predominates only in prostatic fluid; (b) 78, 55, 50 and 43 kDa subunits in prostatic tissue cytosol and (c) 195, 170, 135, 116 and 95 kDa subunits present in the particulate fractions of prostatic tissue. Immunoblotting using specific rabbit antibodies revealed the 50 kDa band to be prostatic acid phosphatase and the 38 kDa band to be prostate-specific antigen. Interestingly, antibodies directed toward prostatic acid phosphatase were found to cross-react with the 43 kDa band. Fractionation on sucrose gradients showed that several of these particulate glycoproteins were associated with a vesicle fraction enriched in adenylate cyclase activity, implying that they are plasma membrane glycoproteins. Comparison of soluble and particulate fractions of normal and cancerous tissue homogenates was made by densitometric scanning of autoradiograms of lectin-loaded gels. Similar relative intensities of lectin-binding were obtained for corresponding proteins in normal and cancerous tissue fractions. Also, immunoblotting showed no differences in prostatic acid phosphatase or prostate-specific antigen between normal and cancerous soluble homogenate fractions. Our results suggest that major lectin-binding proteins are conserved in the transition from normal to cancerous tissue. These results may be useful in developing a multiple-marker profile of metastatic prostate cancer and for the design of imaging agents, such as monoclonal antibodies, to prominent soluble and particulate prostate glycoproteins.

  8. Burn injury reveals altered phenotype in mannan-binding lectin-deficient mice

    DEFF Research Database (Denmark)

    Møller-Kristensen, Mette; Hamblin, Michael R; Thiel, Steffen

    2007-01-01

    Burn injury destroys skin, the second largest innate immune organ in the body, and triggers chaotic immune and inflammatory responses. The pattern recognition molecule, mannan-binding lectin (MBL), plays an important role in the first-line host defense against infectious agents. MBL initiates...... the lectin complement pathway and acts as an opsonin. Recent studies suggest that MBL also modulates inflammatory responses. We report that local responses after burn in MBL null mice differ from those found in wild-type (WT) mice in the following important biological markers: spontaneous eschar separation......, thinned epidermis and dermis, upregulation of soluble factors including cytokines, chemokines, cell adhesion molecules, a growth factor-binding protein, and matrix metalloproteinases. Mice lacking C1q, C4, or C3 did not show the lack of eschar separation seen in MBL null-burn phenotype. These findings...

  9. Exposed and hidden lectin-binding epitopes at the surface of Borrelia burgdorferi

    Czech Academy of Sciences Publication Activity Database

    Stoitsova, S. R.; Grubhoffer, Libor; Nebesářová, Jana

    2003-01-01

    Roč. 48, č. 5 (2003), s. 654-658 ISSN 0015-5632 R&D Projects: GA AV ČR IAA6022001 Grant - others:National Research Council at the Ministry of Education and Science(BG) K-709/97 Institutional research plan: CEZ:AV0Z6022909 Keywords : Borrelia burgdorferi * lectin-binding epitopes Subject RIV: EE - Microbiology, Virology Impact factor: 0.857, year: 2003

  10. The mannan-binding lectin pathway of complement activation: biology and disease association

    DEFF Research Database (Denmark)

    Petersen, Steen Vang; Thiel, S; Jensenius, J C

    2001-01-01

    Mannan-binding lectin (MBL) is a plasma protein found in association with several serine proteases (MASPs) forming the MBL complex. MBL recognises carbohydrate structures arranged in a particular geometry, such as those found on the surface of micro-organisms. When bound to e.g. bacteria the MBL...... as an initiator of the host response against potential pathogenic micro-organisms. Udgivelsesdato: 2001-Aug...

  11. [The significance of Ulex europaeus agglutinin I lectin binding fibers in various muscular diseases].

    Science.gov (United States)

    Yatabe, K; Hiraguri, M; Sueishi, M; Takeuchi, M; Nonaka, I; Kawai, M

    1998-05-01

    In the present study, we have reported that Ulex europaeus agglutinin I (UEA I) lectin labeled muscle fibers in distal myopathy with rimmed vacuole formation (DMRV). UEA I binding to muscle fibers was also observed in a small number of biopsies with inflammatory myopathy, but not in other diseases, including neurogenic muscular atrophies and muscular dystrophies. In order to elucidate the relationship between this UEA I binding, rimmed vacuole formation and active autophagocytosis, we examined the UEA I binding fibers in other myopathies which frequently showed rimmed vacuoles, including adult onset acid maltase deficiency, oculo-pharyngo-distal type myopathy and oculopharyngeal muscular dystrophy. No UEA I lectin labeling fiber was observed in the diseases examined. We then studied UEA I binding behavior on 70 biopsies of inflammatory myopathy to characterize the clinical features of UEA I binding positive patients. UEA I binding fibers were observed in 3 of 28 patients (11%) with other collagen diseases, 11 of 36 (31%) without these disorders, and 2 of 6 (33%) with inclusion body myositis. There were no common clinical histories, complications or laboratory findings among the UEA I binding positive patients. In conclusion, a common process may exist between the muscle fiber degeneration in DMRV and subgroups of inflammatory myopathy patients, but the basic mechanism remains to be elucidated.

  12. Carbohydrate/glycan-binding specificity of legume lectins in respect to their proposed biological functions

    Directory of Open Access Journals (Sweden)

    Márcio Viana Ramos

    2000-01-01

    Full Text Available The lectins, proteins which specifically recognize carbohydrate moieties, have been extensively studied in many biochemical and structural aspects in order to establish the molecular basis of this non-catalytic event. On the other hand, their clinical and agricultural potentials have been growing fast. Although lectins, mainly those from legume plants, had been investigated for biological properties, studies about the physiological functions of lectins are scarce in literature. Therefore, despite the accumulated data on lectins (as proteins, the role played by these signalizing molecules is poorly discussed. In the light of our accumulated results on legume lectins, specially those obtained from plants belonging to the Diocleinae sub-tribe and available data in literature, we discuss here the main hypothesis of their functions according to their carbohydrate/glycan-binding specificity.As lectinas, proteinas que especificamente reconhecem estruturas que contém carboidratos, têm sido extensivamente estudadas em muitos aspectos bioquímicos e estruturais, objetivando estabelecer as bases moleculares deste evento não-catalítico. Por outro lado, os potenciais clínicos e agriculturais destas proteínas têm crescido rapidamente. Embora as lectinas, principalmente aquelas de legumes tenham sido bastante investigadas em suas propriedades biológicas, estudos sobre as funcões fisiológicas de lectinas são escassos na literatura. Além disto, a despeito da quantidade de dados acumulados sobre lectinas (como proteínas, o papel desempenhado por estas moléculas de sinalização é pobremente discutido. Valendo-se de nossos estudos sobre lectinas de leguminosas, principalmente da sub-tribo Diocleinae, e outros dados presentes na literatura, discutimos aqui, as principais hipóteses de suas funções com base na especificidade por carboidratos e glicanos complexos.

  13. Complement-mediated neutralization of dengue virus requires mannose-binding lectin

    DEFF Research Database (Denmark)

    Avirutnan, Panisadee; Hauhart, Richard E; Marovich, Mary A

    2011-01-01

    -dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains...... with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis. IMPORTANCE Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue...... hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation...

  14. Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

    Science.gov (United States)

    Clark, M A; Jepson, M A; Simmons, N L; Hirst, B H

    1995-12-01

    The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

  15. Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

    Science.gov (United States)

    Rao, V. S. R.; Biswas, Margaret; Mukhopadhyay, Chaitali; Balaji, P. V.

    1989-03-01

    The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands. The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β- L-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α- D-glucopyranoside and methyl-2,3-dimethyl-α- D-glucopyranoside which explain well the available experimental data in solution.

  16. Mannose-binding Lectin and the Risk of HIV Transmission and Disease Progression in Children A Systematic Review

    NARCIS (Netherlands)

    Israëls, Joël; Scherpbier, Henriette J.; Frakking, Florine N. J.; van de Wetering, Marianne D.; Kremer, Leontien C. M.; Kuijpers, Taco W.

    2012-01-01

    Background: Mannose-binding lectin (MBL) can activate the complement system by binding to carbohydrates, such as those presented on the HIV virion surface. It is unclear whether genetically determined MBL deficiency is related to vertical HIV transmission and disease progression in HIV-infected

  17. Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction.

    Directory of Open Access Journals (Sweden)

    Shushimita Shushimita

    Full Text Available Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI in mice. We recently showed that Mannan-binding lectin (MBL, the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake or three days of water only fasting on MBL in 10-12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different.

  18. Mannose-binding lectin-2 genotypes and recurrent late pregnancy losses

    DEFF Research Database (Denmark)

    Christiansen, Ole B; Nielsen, Henriette S; Lund, Marie

    2008-01-01

    maternal rather than fetal causes are likely to play a stronger role than in early recurrent miscarriage. METHODS: We identified 75 patients with at least two late losses of pregnancies with apparently normal fetuses between gestational week 14 and 30 among patients with recurrent pregnancy losses referred......BACKGROUND: Low levels of mannose-binding lectin (MBL) predispose to various infectious and inflammatory disorders and have been reported to be associated with recurrent early miscarriages. Recurrent late pregnancy losses (RLPL) in the second trimester is a rare but devastating syndrome where...... is strongly associated with idiopathic RLPL. This may point towards a role for excessive inflammatory disturbances as a cause of the syndrome....

  19. Cyborg lectins: novel leguminous lectins with unique specificities.

    Science.gov (United States)

    Yamamoto, K; Maruyama, I N; Osawa, T

    2000-01-01

    Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.

  20. Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC

    Directory of Open Access Journals (Sweden)

    Markus Roucka

    2016-12-01

    Full Text Available Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC-optimized variant (MB314. MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL, Pisum sativum agglutinin (PSA, Lens culinaris agglutinin (LCA, and Aleuria aurantia lectin (AAL were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody—whose affinity is mediated through core fucosylation—and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

  1. A chitin-binding lectin from Moringa oleifera seeds (WSMoL) impairs the digestive physiology of the Mediterranean flour larvae, Anagasta kuehniella.

    Science.gov (United States)

    de Oliveira, Caio Fernando Ramalho; de Moura, Maiara Celine; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; Coelho, Luana Cassandra Breitenbach Barroso; Macedo, Maria Lígia Rodrigues

    2017-10-01

    Biotechnological techniques allow the investigation of alternatives to outdated chemical insecticides for crop protection; some investigations have focused on the identification of molecules tailored from nature for this purpose. We, herein, describe the negative effects of water-soluble lectin from Moringa oleifera seeds (WSMoL) on Anagasta kuehniella development. The chitin-binding lectin, WSMoL, impaired the larval weight gain by 50% and affected the activity of the pest's major digestive enzymes. The commitment of the digestive process became evident after controlled digestion studies, where the capacity of protein digestion was compromised by >90%. Upon acute exposure, the lectin was not resistant to digestion; however, chronic ingestion of WSMoL was able to reverse this feature. Thus, we show that resistance to digestion may not be a prerequisite for a lectin's ability to exert negative effects on larval physiology. The mechanism of action of WSMoL involves binding to chitin with possible disruption to the peritrophic membrane, causing disorder between the endo- and ectoperitrophic spaces. Additionally, results suggest that WSMoL may trigger apoptosis in gut cells, leading to the lower enzymatic activity observed in WSMoL-fed larvae. Although assays employing an artificial diet did not demonstrate effects of WSMoL on A. kuehniella mortality, this lectin may hold potential for exerting insecticide effects on other pest insects, as well for use in other experimental approaches, such as WSMoL-expressing plants. Moreover, the use of WSMoL with other biotechnological tools, such as 'pyramid' crops, may represent a strategy for delaying the evolution of pest resistance to transgenic crops, since its multiple site targets could act in synergism with other insecticide compounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Investigation on interaction of Achatinin, a 9-O-acetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails.

    Science.gov (United States)

    Biswas, C; Sinha, D; Mandal, C

    2000-01-01

    Achatinin, a 9-O-acetyl sialic acid (9-O-AcSA) binding lectin, has been demonstrated to be synthesized in amoebocytes of Achatina fulica snails. This lectin was affinity-purified from Achatina amoebocytes lysate (AAL); it appeared as a single band on native polyacrylamide gel electrophoresis (PAGE) and showed 16 identical subunits of M.W. 15 kDa on sodium dodecyl sulphate (SDS)-PAGE. It was found to be homologous with an earlier reported lectin, Achatinin-H, derived from hemolymph of A. fulica snails (Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achantia fulica. Carbohydr. Res., 268, 115-125). Homology between both lectins was confirmed by their similar electrophoretic mobilities, carbohydrate specificity and cross reactivity on immunodiffusion. Achatinin showed in vitro calcium dependent binding to two 9-O-acetylated sialoglyoconjugates (9-O-AcSG) on lipopolysaccharide (LPS) (Escherichia coli 055: B5) of M.W. 40 kDa and 27.5 kDa, which was abolished following de-O-acetylation. Based on the previously defined narrow sugar specificity of Achatinin towards 9-O-AcSAalpha2-->6GalNAc [Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achatina fulica. Carbohydr. Res., 268, 115-125], we conclude that LPS contains this lectinogenic epitope at the terminal sugar moiety. The Achatinin-mediated hemagglutination inhibition of rabbit erythrocytes by LPS further confirmed it. The lectin exhibited bacteriostatic effect on Gram-negative bacteria E. coli, DH5alpha and C600. AAL was earlier reported to undergo coagulation in presence of pg level of LPS (Biswas, C., Mandal, C., 1999. The role of amoebocytes in the endotoxin-mediated coagulation in the innate immunity of Achatina fulica snail, Scand. J. Immunol. 49, 131-138). We now demonstrate that Achatinin participates in LPS-mediated coagulation of AAL as indicated by enhanced release of Achatinin from

  3. Acid phosphatase from stored Poa pratensis caryopses and its ability for binding to lectins

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2014-01-01

    Full Text Available The effect of the storage period of Poa pratensis caryopses on acid phosphatase activity and on the ability of this enzyme to interact with lectins has been studied. It has been shown that after ten years of caryopses storage, the activity of acid phosphatase decreased about 50 per cent, while the content of proteins and carbohydrates did not change. The decrease of enzyme activity during the long period of storage was found only in seeds, but not in chaffs. Acid phosphatase was isolated from caryopses stored one, two, three, five and ten years. The enzyme showed the ability to bind to immoblized as well as to free conA during the whole period of storage, hut did not react with Wheat Germen Agglutinin (WGA. The activation of acid phosphatase by binding to conA decreased with the length of storage period.

  4. Binding of peanut lectin to germinal-centre cells: a marker for B-cell subsets of follicular lymphoma?

    OpenAIRE

    Rose, M. L.; Habeshaw, J. A.; Kennedy, R.; Sloane, J.; Wiltshaw, E.; Davies, A. J.

    1981-01-01

    The binding of horseradish-peroxidase-labelled peanut lectin (HRP-PNL) to cryostat sections of tonsil, lymphoma lymph nodes, reactive lymph nodes and miscellaneous tumours demonstrated that PNL binds selectively to lymphocytes in germinal centres. Lymph nodes from 21 patients with non-Hodgkin's lymphomas were phenotyped as cell suspensions for PNL binding, and the following surface markers: E rosetting, C3d, SIg, OK markers of T-cell subsets, Ig heavy-chain and light-chain classes. There was ...

  5. Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

    Science.gov (United States)

    Basu, S; Mandal, C; Allen, A K

    1988-01-01

    A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin. Images Fig. 3. PMID:3140796

  6. A simple procedure for the isolation of L-fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus.

    Science.gov (United States)

    Allen, H J; Johnson, E A

    1977-10-01

    L-Fucose-binding lectins from Ulex europeaus and Lotus tetragonolobus were isolated by affinity chromatography on columns of L-fucose-Sepharose 6B. L-Fucose was coupled to Sepharose 6B after divinyl sulfone-activation of the gel to give an affinity adsorbent capable of binding more than 1.2 mg of Ulex lextin/ml of gel, which could then be eluted with 0.1M or 0.05M L-fucose. Analysis of the isolated lectins by hemagglutination assay, by gel filtration, and polyacrylamide disc-electrophoresis revealed the presence of isolectins, or aggregated species, or both. The apparent mol. wt. of the major lectin fraction from Lotus was 35000 when determined on Sephadex G-200 or Ultrogel AcA 34. In contrast, the apparent mol. wt. of the major lectin fraction from Ulex was 68 000 when chromatographed on Sephadex G-200 and 45 000 when chromatographed on Ultrogel AcA 34. The yields of lectins were 4.5 mg/100 g of Ulex seeds and 394 mg/100 g of Lotus seeds.

  7. Presence of the variant mannose-binding lectin alleles associated with slower progression to AIDS. Amsterdam Cohort Study

    NARCIS (Netherlands)

    Maas, J.; de Roda Husman, A. M.; Brouwer, M.; Krol, A.; Coutinho, R.; Keet, I.; van Leeuwen, R.; Schuitemaker, H.

    1998-01-01

    OBJECTIVE: To examine the association between mannose-binding lectin (MBL) polymorphism and progression to AIDS and death in HIV-1 infection. DESIGN AND METHODS: In 131 HIV-1-infected homosexual seroconverters, survival analyses were performed to determine both the association between MBL genotype

  8. L-rhamnose-binding lectins (RBLs) in Nile tilapia, Oreochromis niloticus: characterization and expression profiling in mucosal tissues

    Science.gov (United States)

    Rhamnose-binding lectins (RBLs) are crucial elements associated with innate immune responses to infections and have been characterized from a variety of teleost fishes. Our previous work highlighted a major role of a RBL (IpRBL1a) in mediating F. columnare adhesion and IpRBL1a showed higher expressi...

  9. Mannose-binding lectin gene, MBL2, polymorphisms are not associated with susceptibility to invasive pneumococcal disease in children

    DEFF Research Database (Denmark)

    Lundbo, Lene Fogt; Harboe, Zitta Barrella; Clausen, Louise Nygaard

    2014-01-01

    BACKGROUND: Most children are transiently colonized with Streptococcus pneumoniae, but very few develop invasive pneumococcal disease (IPD). Host genetic variation of innate immunity may predispose to IPD. We investigated the effect of genetic variation in the mannose-binding lectin gene, MBL2......, on susceptibility and disease severity of IPD in previously healthy children aged

  10. Wild boars from Sweden, Austria, the Czech Republic and Japan possess intact mannose-binding lectin 2 (MBL2) genes

    DEFF Research Database (Denmark)

    Bergmann, Ingrid-Maria; OkumuRA, N; Uenishi, H

    2015-01-01

    The two-nucleotide deletion recently detected in the mannose-binding lectin 2 gene in purebred and crossbred domestic pigs was not found among 68 wild boars representing 4 populations from Europe and Asia. This suggests that the deletion is a result of breeding and/or genetic drift/bottle necks....

  11. Lectin binding assays for in-process monitoring of sialylation in protein production.

    Science.gov (United States)

    Xu, Weiduan; Chen, Jianmin; Yamasaki, Glenn; Murphy, John E; Mei, Baisong

    2010-07-01

    Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galbeta1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(beta1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galbeta1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galbeta1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.

  12. Lectins binding during alloxan-induced diabetes in rat soleus muscle

    African Journals Online (AJOL)

    Membrane structural changes of soleus muscle of alloxan-diabetic rats were detected with a panel of six biotinylated lectins. Samples of muscles were obtained from normal and diabetic rats. The biotinylated lectins in staining were detected by avidin-peroxidase complex. Lectin stainning of soleus muscle cryostat sections ...

  13. Influence of chicken serum mannose-binding lectin levels on the immune response towards Escherichia coli

    DEFF Research Database (Denmark)

    Norup, L R; Dalgaard, T; Friggens, N

    2009-01-01

    This study aimed to investigate the effect of mannose-binding lectin (MBL) on infections with Escherichia coli in chickens. Initially, the basic levels of MBL in 4 different lines of layer chickens, namely ISA Brown, Lohmann Selected Leghorn, Lohmann Braun, and Hellevad, were investigated....... This investigation revealed a 2-to 3-fold difference in the basic levels of MBL in serum between some of these commercial lines. Furthermore, the ontogeny of the basic level of MBL in serum of an experimental chicken line was investigated. The level of MBL was very stabile for long periods, with an elevation at 5...... to 7 wk of age. Another elevation in MBL level started around 18 to 19 wk of age and stayed elevated at least until 38 wk of age. In this study, it was hypothesized that chickens with high levels of MBL (H-type) may be less prone to disease caused by E. coli infection than chickens with low levels...

  14. Increased activity of the mannan-binding lectin complement activation pathway in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Ytting, H; Jensenius, Jens Christian; Christensen, I J

    2004-01-01

    BACKGROUND: Postoperative bacterial infectious complications are frequent in patients with colorectal cancer (CRC), with subsequent increased recurrence rates and poor prognosis. Deficiency of the mannan-binding lectin (MBL) complement activation pathway may cause increased risk of infection......: Serum MBL concentrations and MBL/MASP activity were determined using immunofluorometric assays. The levels are presented as the median, inter-quartile range and range. RESULTS: Serum MBL levels were significantly (P cancer (1384 (400-2188) ng/mL) (median...... in the colon or rectum, and disease stages according to Dukes' classification. No statistical difference (P=0.20) in frequency of MBL deficiency was found between the patients (20%) and the donors (27%). CONCLUSIONS: Overall, the MBL complement activation pathway is significantly increased in patients...

  15. The pattern-recognition molecule mannan-binding lectin (MBL) in the pathophysiology of diabetic nephropathy

    DEFF Research Database (Denmark)

    Axelgaard, Esben; Thiel, Steffen; Hansen, Troels Krarup

    to carbohydrates of both specific type and density, which thus provides sufficient binding avidity. The character of MBL binding-sites on host cells remain unknown, but it is speculated that altered protein glycation in diabetes permits MBL binding. Based on new studies using MBL/double knockout C57bl/6j mice, we...

  16. Incident microalbuminuria and complement factor mannan-binding lectin-associated protein 19 in people with newly diagnosed type 1 diabetes

    DEFF Research Database (Denmark)

    Ostergaard, J A; Thiel, S; Hoffmann-Petersen, I T

    2017-01-01

    BACKGROUND: Evidence links the lectin pathway of complement activation to diabetic kidney disease. Upon carbohydrate-recognition by pattern-recognition molecules, e.g., mannan-binding lectin (MBL), the MBL-associated serine protease (MASP-2) is activated and initiates the complement cascade. The ...

  17. Circulating mannan-binding lectin, M-, L-, H-ficolin and collectin-liver-1 levels in patients with acute liver failure

    DEFF Research Database (Denmark)

    Laursen, Tea Lund; Sandahl, Thomas D; Støy, Sidsel

    2015-01-01

    BACKGROUND & AIMS: The complement system is activated in liver diseases including acute liver failure (ALF); however, the role of the lectin pathway of complement has scarcely been investigated in ALF. The pathway is initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL),...

  18. Binding properties of a blood group Le(a+) active sialoglycoprotein, purified from human ovarian cyst, with applied lectins.

    Science.gov (United States)

    Wu, A M; WU, J H; Watkins, W M; Chen, C P; Tsai, M C

    1996-06-07

    Studies on the structures and binding properties of the glycoproteins, purified from human ovarian cyst fluids, will aid the understanding of the carbohydrate alterations occurring during the biosynthesis of blood group antigens and neoplasm formation. These glycoproteins can also serve as important biological materials to study blood group A, B, H, Le(a), Le(b), Le(x), Le(y), T and Tn determinants, precursor type I and II sequences and cold agglutinin I and i epitopes. In this study, the binding property of a cyst glycoprotein from a human blood group Le(a+) nonsecretor individual, that contains an unusually high amount (18%) of sialic acid (HOC 350) was characterized by quantitative precipitin assay with a panel of lectins exhibiting a broad range of carbohydrate-binding specificities. Native HOC 350 reacted well only with three out of nineteen lectins tested. It precipitated about 80% of Ricinus communis (RCA1), 50% of Triticum vulgaris (WGA) and 37% of Bauhinia purpurea aba (BPA) agglutinins, respectively. However, its asialo product had dramatically enhanced reactivity and reacted well with many I/II (Gal beta1 --> 3/4GcNAc), T(Gal beta1 --> 3GalNAc) and Tn(GaNIAc alphaI --> Ser/Thr) active lectins. It bound best to Jacalin, BPA, and abrin-a and completely precipitated all the lectins added. Asialo-HOC 350 also reacted strongly with Wistaria floribunda, Abrus precatorius agglutinin, ricin and RCA1 and precipitated over 75% of the lectin nitrogen added, and moderately with Arachis hypogaea, Maclura pomifera, WGA, Vicia viosa-B4, Codium fragile tomentosoides and Ulex europaeus-II. But native HOC 350 and its asialo product reacted not at all or poorly with Dolichos biflorus, Helix pomatia, Lotus tetra-gonolobus, Ulex europaeus-I, Lens culinaris lectins and Con A. The lectin-glycoform interactions through bioactive sugars were confirmed by precipitin inhibition assay. Mapping the precipitation profiles of the interactions have led to the conclusion that HOC 350

  19. Targeting the Cryptococcus neoformans var. grubii Cell Wall Using Lectins: Study of the Carbohydrate-Binding Domain

    Directory of Open Access Journals (Sweden)

    Pamella de Brito Ximenes

    2015-02-01

    Full Text Available Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis in immunosuppressed patients. Understanding cell wall glycoproteins using lectins is of medical interest and can contribute to specific therapy. The aim of this study was to evaluate the carbohydrates on the cell wall of Cryptococcus neoformans var. grubii clinical isolates, using a fluorescein isothiocyanate-lectin binding protocol. Thirty yeast strains stocked in the culture collection were cultivated for 2 days at 30 °C with shaking. Cells were obtained by centrifugation, washed in phosphate-buffered saline, and a suspension of 107 cells/mL was obtained. To determine the binding profile of lectins, concanavalin A (Con A, wheat germ agglutinin (WGA, Ulex europaeus agglutinin I (UEA-I, and peanut agglutinin (PNA conjugated to fluorescein were used. All the tested clinical isolates of Cryptococcus neoformans var. grubii were intensely stained by WGA, moderately stained by Con A, and weakly stained by PNA and UEA-I. Thus, Cryptococcus can be detected in clinical specimens such as blood and cerebrospinal fluid using the fluorescent lectin WGA, which may be considered as an option for detection in cases of suspected cryptococcosis with low laboratory sensitivity. Future applications may be developed using this basic tool.

  20. Mannose binding lectin (MBL levels predict lung function decline in severe asthma

    Directory of Open Access Journals (Sweden)

    Ilonka. H. van Veen

    2006-12-01

    Full Text Available There is increasing evidence that activation of the complement system in asthma contributes to ongoing inflammation, tissue damage and airway remodeling. Mannose binding lectin (MBL is a pattern recognition molecule that serves as the key mediator of the lectin pathway of complement activation. MBL levels are genetically determined and vary widely amongst individuals. In the present study we hypothesized that high MBL levels in asthma are associated with increased loss of lung function over time, as a consequence of inflammatory tissue damage. We measured serum MBL levels by ELISA in 68 patients with severe asthma and prospectively determined the change in post-bronchodilator (pb FEV1 over a mean period of 5.7 years. The relationship between MBL and change in pbFEV1 (FEV1 was analysed using (multiple regression analysis and corrected for possible confounders (age, sex, age of onset, asthma duration, and pbFEV1. The median (range MBL level was 332 (10.8-3587 ng·ml–1. MBL was significantly associated with FEV1 (p<0.04. Patients with a high MBL level (332 ng·ml–1 had an increased risk of lung function decline compared to those with low MBL levels (OR (CI: 3.16 (1.14-8.79, p = 0.027; the excess decline being 42 ml·yr–1 (p = 0.01. We conclude that a high MBL level is associated with an increased decline in lung function in patients with severe asthma. MBL might provide a clue towards better understanding of the pathophysiology of ongoing inflammation and subsequent decline in lung function of patients with severe asthma.

  1. Genetically determined serum levels of mannose-binding lectin correlate negatively with common carotid intima-media thickness in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Troelsen, Lone N; Garred, Peter; Christiansen, Buris

    2010-01-01

    Patients with systemic lupus erythematosus (SLE) have excess cardiovascular morbidity and mortality due to accelerated atherosclerosis that cannot be attributed to traditional cardiovascular risk factors alone. Variant alleles of the mannose-binding lectin gene (MBL2) causing low serum...

  2. Evaluation of lectin pathway activity and mannan-binding lectin levels in the course of pregnancy complicated by diabetes type 1, based on the genetic background.

    Science.gov (United States)

    Pertyńska Marczewska, Magdalena; Cedzyński, Maciej; Swierzko, Anna; Szala, Agnieszka; Sobczak, Małgorzata; Cypryk, Katarzyna; Wilczyński, Jan

    2009-01-01

    There are numerous indications that either mannan-binding lectin (MBL) deficiency or its excessive activity are associated with adverse pregnancy outcomes. High MBL concentrations and corresponding MBL2 genotypes were shown to be associated with microvascular complications in type 1 diabetes. The aim of this study was to evaluate levels of MBL and MBL-dependent activity of the lectin pathway (LP) of complement in the course of pregnancy in diabetic mothers, based on genetic background. These parameters were determined in samples from healthy non-pregnant (control), diabetic non-pregnant, healthy pregnant, and pregnant diabetic women. No significant differences in median MBL levels or LP activities were found in any study group compared to the control. However, statistically significant differences in MBL levels were noted during pregnancy between the 1st and 3rd trimesters in both healthy controls and pregnant diabetics. With regard to LP values, similar trends were evident, but statistically significant results were obtained only in the healthy pregnant group. When data analysis was confined to patients carrying the A/A (wild-type) MBL2 genotype, an increase in MBL level during pregnancy (in both healthy and diabetic pregnant women) was still observed. Similarly, LP activity increased during both healthy and diabetic pregnancies, significantly so for the former. Diabetes, an autoimmune disease, is a serious complication of pregnancy. Therefore, determination of MBL status might be beneficial in identifying type 1 diabetic patients who are at increased risk of developing both vascular complications and poor pregnancy outcomes.

  3. Deposition of mannose-binding lectin and ficolins and activation of the lectin pathway of complement on the surface of polyurethane tubing used for cardiopulmonary bypass.

    Science.gov (United States)

    Eppa, Łukasz; Pągowska-Klimek, Izabela; Świerzko, Anna S; Moll, Maciej; Krajewski, Wojciech R; Cedzyński, Maciej

    2018-04-01

    The artificial surface used for cardiopulmonary bypass (CPB) is a crucial factor activating the complement system and thus contributing to the generation of a systemic inflammatory response. The activation of classical and alternative pathways on this artificial surface is well known. In contrast, lectin pathway (LP) activation has not been fully investigated, although noted during CPB in several studies. Moreover, we have recently proved the contribution of the LP to the generation of the systemic inflammatory response syndrome after pediatric cardiac surgery. The aim of this study was to assess LP-mediated complement activation on the surface of polyurethane CPB circuit tubing (noncoated Chalice ® ), used for CPB procedures in children with congenital heart disease. We found deposition of mannose-binding lectin, ficolin-1, -2, and -3 on the surface of unused tubing and on tubing used for CPB from a small minority of patients. Furthermore, we observed deposition of complement C4 activation products on tubing used for CPB and previously unused tubing after incubation with normal serum. The latter finding indicates LP activation in vitro on the polyurethane surface. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1202-1208, 2018. © 2017 Wiley Periodicals, Inc.

  4. Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose-Binding Lectin, on HeLa Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Carla Oliveira

    2011-01-01

    Full Text Available Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

  5. Distribution of Glycan Motifs at the Surface of Midgut Cells in the Cotton Leafworm (Spodoptera littoralis Demonstrated by Lectin Binding

    Directory of Open Access Journals (Sweden)

    Tomasz Walski

    2017-12-01

    Full Text Available Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA, GalNAc/Gal (RSA and SSA, GlcNAc (WGA and Nictaba and Neu5Ac(α-2,6Gal/GalNAc (SNA-I], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and

  6. Purification of a novel chitin-binding lectin with antimicrobial and antibiofilm activities from a bangladeshi cultivar of potato (Solanum tuberosum).

    Science.gov (United States)

    Hasan, Imtiaj; Ozeki, Yasuhiro; Kabir, Syed Rashel

    2014-04-01

    A new chitin-binding lectin was purified from a Bangladeshi cultivar 'Deshi' of potato (Solanum tuberosum L.) through anion-exchange and affinity chromatographies using a chitin column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the molecular mass of the lectin as 20,000 Daltons. This molecular mass was almost half of the molecular masses of chitin-binding lectins derived from other potatoes. The lectin showed both bactericidal and growth-inhibiting activities against Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli, Salmonella enteritidis and Shigella boydii) pathogenic bacteria. It also showed antifungal activity against Rhizopus spp., Penicillium spp. and Aspergillus niger. Biofilm produced by the bacterium Pseudomonas aeruginosa was dose-dependently reduced by 5-20% in 24 h after administration of the lectin, which was attributed to the glycan-binding property of the lectin having affinity to GlcNAc polymers. It was the first observation that any potato lectin prevented biofilm formation by P. aeruginosa and, therefore, could have possible applications in clinical microbiology and biomedical science.

  7. Mannose-Binding Lectin Levels and Carotid Intima-Media Thickness in Type 2 Diabetic Patients

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    Miklós Káplár

    2016-01-01

    Full Text Available Introduction. Mannose-binding lectin (MBL activates complement system and has been suggested to play a role in vascular complications in diabetics. Carotid intima-media thickness (cIMT detects subclinical atherosclerosis. We evaluated the association of MBL and IMT in type 2 diabetic (T2DM patients. Methods. Serum MBL levels and cIMT were measured in a total of 103 diabetics and in 98 age-matched healthy controls. Results. There was no significant difference in MBL level in T2DM versus controls. As expected, IMT was significantly higher in T2DM patients than in controls (P=0.001. In T2DM, the lowest cIMT was seen in patients with normal MBL level (500–1000 while cIMT continuously increased with both high MBL and absolute MBL deficiency states. This was especially significant in high MBL versus normal MBL T2DM patients (P=0.002. According to multiple regression analysis the main predictors of IMT in T2DM are age (P<0.003, ApoA level (P=0.023, and the MBL (P=0.036. Conclusions. Our results suggest a dual role of MBL as a risk factor for cIMT in T2DM. MBL may also be used as a marker of macrovascular disease, as both low and high levels indicate the susceptibility for atherosclerosis in T2DM.

  8. Mannose-Binding Lectin and Toll-Like Receptor Polymorphisms and Chagas Disease in Chile

    Science.gov (United States)

    Zulantay, Inés; Danquah, Ina; Hamann, Lutz; Schumann, Ralf R.; Apt, Werner; Mockenhaupt, Frank P.

    2012-01-01

    Mannose-binding lectin (MBL) and Toll-like receptor (TLR) polymorphisms may influence susceptibility and manifestation of Trypanosoma cruzi infection. In northern Chile, we examined 61 asymptomatic patients with chronic Chagas disease (CD), 64 patients with chronic Chagas cardiomyopathy (CCC), and 45 healthy individuals. Low-producer MBL2*B genotypes were more common in CD patients (48%) than healthy individuals (31%; adjusted odds ratio = 2.3, 95% confidence interval = 1.01–5.4, P = 0.047) but did not differ with manifestation. In contrast, the heterozygous Toll-like receptor 4 (TLR4)-deficiency genotype D299G/T399I occurred more frequently in asymptomatic (14.8%) than CCC patients (3.1%; P = 0.02). TLR1-I602S, TLR2-R753Q, TLR6-S249P, and MAL/TIRAP-S180L did not associate with CD or CCC. These findings support the complement system to be involved in defense against Trypanosoma cruzi infection and indicate that curbed TLR4 activation might be beneficial in preventing CCC. PMID:22302853

  9. Mannose-binding lectin genotypes: lack of association with susceptibility to thoracic empyema

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    Moore Catrin E

    2010-01-01

    Full Text Available Abstract Background The role of the innate immune protein mannose-binding lectin (MBL in host defence against severe respiratory infection remains controversial. Thoracic empyema is a suppurative lung infection that arises as a major complication of pneumonia and is associated with a significant mortality. Although the pathogenesis of thoracic empyema is poorly understood, genetic susceptibility loci for this condition have recently been identified. The possible role of MBL genotypic deficiency in susceptibility to thoracic empyema has not previously been reported. Methods To investigate this further we compared the frequencies of the six functional MBL polymorphisms in 170 European individuals with thoracic empyema and 225 healthy control individuals. Results No overall association was observed between MBL genotypic deficiency and susceptibility to thoracic empyema (2 × 2 Chi square = 0.02, P = 0.87. Furthermore, no association was seen between MBL deficiency and susceptibility to the Gram-positive or pneumococcal empyema subgroups. MBL genotypic deficiency did not associate with progression to death or requirement for surgery. Conclusions Our results suggest that MBL genotypic deficiency does not associate with susceptibility to thoracic empyema in humans.

  10. A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.

    Science.gov (United States)

    Kim, Seonghun

    2018-02-01

    Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Changes in the levels of mannan-binding lectin and ficolins during head-down tilted bed rest.

    Science.gov (United States)

    Kelsen, Jens; Sandahl, Thomas D; Storm, Line; Frings-Meuthen, Petra; Dahlerup, Jens F; Thiel, Steffen

    2014-08-01

    Spaceflight studies and ground-based analogues of microgravity indicate a weakening of human immunity. Mannan-binding lectin (MBL) and H-, L-, and M-ficolin together constitute the lectin pathway and mediate the clearance of pathogens through complement activation. We hypothesized that simulated microgravity may weaken human innate immune functions and studied the impact of 6° head-down tilted bed rest (HDT) for 21 d on MBL and ficolin levels. Within a 6-mo period, seven men underwent two periods of HDT. Blood samples were analyzed for MBL, H-, L-, and M-ficolin, mannose-binding lectin-associated protein of 44 kDa (MAp44), and collectin liver 1 (CL-L1) by time-resolved immunofluorometric assays (TRIFMA). We observed well-defined individual preintervention levels of MBL and ficolins. Remarkably similar intraindividual changes occurred for MBL and MBL levels decreased (mean 282 ng · ml⁻¹) in the recovery phase. Conversely, CL-L1, a protein with MBL-like properties, increased (mean 102 ng · ml⁻¹) during the recovery phase. M-ficolin increased (mean 79 ng · ml⁻¹) within the first 2 d of HDT, followed by a decrease (mean 112 ng · ml⁻¹) during the recovery phase. L-ficolin increased (mean 304 ng · ml⁻¹) during HDT, while H-ficolin was essentially unaffected. MAp44, a down-regulator of the lectin pathway, decreased initially (mean 78 ng · ml⁻¹) in the recovery phase followed by an increase (mean 131 ng · ml⁻¹). Alterations in MBL and ficolin levels were modest and with our current knowledge do not lead to overt immunodeficiency. Pronounced changes occurred when the subjects resumed the upright position. In selected individuals, these changes appear to be a conserved response to HDT.

  12. The Role of Mannose-Binding Lectin Serum Level in Tubotympanic Chronic Suppurative Otitis Media

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    Anton Budhi Darmawan

    2018-01-01

    Full Text Available Background. Chronic suppurative otitis media (CSOM is a common public health problem worldwide and a major cause of hearing impairment especially in developing countries. The role of Mannose-Binding Lectin (MBL, a component of innate immunity, in CSOM has not been studied. The aim of the study was to examine whether MBL deficiency was more frequently present in cases group of tubotympanic CSOM patients rather than healthy subjects. Material and Methods. This was an analytic observational study. Subjects were enrolled in the Otorhinolaryngology Clinic at Margono Soekarjo Hospital, Purwokerto, Indonesia. An independent t-test was used to compare the mean of MBL serum concentration between tubotympanic CSOM subjects and control. Results. From 36 tubotympanic CSOM patients, there were 8 (22.22% patients with MBL deficiency (MBL level < 100 ng/ml, while no deficiency was found in the control group. The mean of MBL level in cases group was 354.88 ng/ml, with the lowest level being 0.001 ng/ml and the highest level 690.24 ng/ml, while in the control group MBL level mean was 376.27 with the lowest level being 188.71 and the highest level 794.54 ng/ml. Conclusion. There was no significant difference of MBL serum level between tubotympanic CSOM and control group. However, the presence of subjects with MBL deficiency in the tubotympanic CSOM group might be considered as playing a role in the tubotympanic CSOM.

  13. Effect of Optimization of Glycaemic Control on Mannan-Binding Lectin in Type 1 Diabetes

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    Gry Høst Dørflinger

    2017-01-01

    Full Text Available Objective. Mannan-binding lectin (MBL concentration in plasma is increased in subjects with type 1 diabetes and associated with increased mortality and risk of diabetic nephropathy. Recent findings show that pancreas transplantation reduces MBL concentration. Whether the increased MBL concentration is reversed by improved glycaemic control remains unknown. We investigated the effects of improved glycaemic control on MBL concentration in patients with type 1 diabetes. Methods. We measured MBL, fructosamine, and HbA1cat baseline and after 6 weeks in 52 type 1 diabetic patients following the change from conventional insulin therapy to insulin pump therapy. Results. After initiation of insulin pump therapy, the total daily insulin dose was significantly reduced (from 51 ± 18 IE/day to 39 ± 13 IE/day, P<0.0001. There was a significant decrease in HbA1c from 8.6% to 7.7% (from 70 mmol/mol to 61 mmol/mol, P<0.0001 and in fructosamine levels (from 356 μmol/L to 311 μmol/L, P<0.0001. MBL levels decreased by 10% from 2165 μg/L (IQR 919–3389 μg/L at baseline to 1928 μ/L (IQR 811–2758 μg/L at follow-up (P=0.005, but MBL change was not significantly correlated with changes in insulin dose, HbA1c, or fructosamine. Conclusions. MBL concentration decreased following the initiation of insulin pump therapy in patients with type 1 diabetes and did not correlate with changes in glycaemic control.

  14. The salivary scavenger and agglutinin (SALSA binds MBL and regulates the lectin pathway of complement in solution and on surfaces

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    Martin eParnov Reichhardt

    2012-07-01

    Full Text Available The scavenger receptor cysteine-rich (SRCR protein SALSA, also known as gp340, salivary agglutinin (SAG and deleted in malignant brain tumor 1 (DMBT1, is a 340 kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A (SP-D and SP-A and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan binding lectin (MBL as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit C. albicans-induced complement activation. Thus, SALSA has a dual complement regulatory function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid-phase. These activities are mediated via a direct interaction with MBL.

  15. Lectins, Interconnecting Proteins with Biotechnological/Pharmacological and Therapeutic Applications

    Directory of Open Access Journals (Sweden)

    Luana Cassandra Breitenbach Barroso Coelho

    2017-01-01

    Full Text Available Lectins are proteins extensively used in biomedical applications with property to recognize carbohydrates through carbohydrate-binding sites, which identify glycans attached to cell surfaces, glycoconjugates, or free sugars, detecting abnormal cells and biomarkers related to diseases. These lectin abilities promoted interesting results in experimental treatments of immunological diseases, wounds, and cancer. Lectins obtained from virus, microorganisms, algae, animals, and plants were reported as modulators and tool markers in vivo and in vitro; these molecules also play a role in the induction of mitosis and immune responses, contributing for resolution of infections and inflammations. Lectins revealed healing effect through induction of reepithelialization and cicatrization of wounds. Some lectins have been efficient agents against virus, fungi, bacteria, and helminths at low concentrations. Lectin-mediated bioadhesion has been an interesting characteristic for development of drug delivery systems. Lectin histochemistry and lectin-based biosensors are useful to detect transformed tissues and biomarkers related to disease occurrence; antitumor lectins reported are promising for cancer therapy. Here, we address lectins from distinct sources with some biological effect and biotechnological potential in the diagnosis and therapeutic of diseases, highlighting many advances in this growing field.

  16. The galactose-binding lectin isolated from Bauhinia bauhinioides Mart seeds inhibits neutrophil rolling and adhesion via primary cytokines.

    Science.gov (United States)

    Girão, Deysen Kerlla Fernandes Bezerra; Cavada, Benildo Sousa; de Freitas Pires, Alana; Martins, Timna Varela; Franco, Álvaro Xavier; Morais, Cecília Mendes; Santiago do Nascimento, Kyria; Delatorre, Plinio; da Silva, Helton Colares; Nagano, Celso Shiniti; Assreuy, Ana Maria Sampaio; Soares, Pedro Marcos Gomes

    2015-05-01

    In this study, the amino acid sequence and anti-inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% - 3 h, 19% - 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF-α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate-binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF-α (68%) and IL1-β (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration by reducing TNF-α and IL1-β levels via the lectin domain. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Global Autorecognition and Activation of Complement by Mannan-Binding Lectin in a Mouse Model of Type 1 Diabetes

    Directory of Open Access Journals (Sweden)

    Esben Axelgaard

    2017-01-01

    Full Text Available Increasing evidence links mannan-binding lectin (MBL to late vascular complications of diabetes. MBL is a complement-activating pattern recognition molecule of the innate immune system that can mediate an inflammation response through activation of the lectin pathway. In two recent animal studies, we have shown that autoreactivity of MBL is increased in the kidney in diabetic nephropathy. We hypothesize that long-term exposure to uncontrolled high blood glucose in diabetes may mediate formation of neoepitopes in several tissues and that MBL is able to recognize these structures and thus activate the lectin pathway. To test this hypothesis, we induced diabetes by injection of low-dose streptozotocin in MBL double-knockout (MBL/DKO mice. Development of diabetes was followed by measurements of blood glucose and urine albumin-to-creatinine ratio. Fluorophore-labelled recombinant MBL was injected intravenously in diabetic and nondiabetic mice followed by ex vivo imaging of several organs. We observed that MBL accumulated in the heart, liver, brain, lung, pancreas, and intestines of diabetic mice. We furthermore detected increased systemic complement activation after administration of MBL, thus indicating MBL-mediated systemic complement activation in these animals. These new findings indicate a global role of MBL during late diabetes-mediated vascular complications in various tissues.

  18. The galactophilic lectin (PA-IL, gene LecA) from Pseudomonas aeruginosa. Its binding requirements and the localization of lectin receptors in various mouse tissues

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Hansen, Axel K; d'Apice, Anthony

    2006-01-01

    . aeruginosa lectin were compared with the results obtained using an isolectin from the legume shrub Griffonia simplicifolia: the GSI-134 isolectin, which is highly specific for glycans terminating in Ga1 alpha 1-R. In the wild-type mice, lectin histochemistry showed a strong capillary reaction in heart...

  19. CancerLectinDB: a database of lectins relevant to cancer.

    Science.gov (United States)

    Damodaran, Deepa; Jeyakani, Justin; Chauhan, Alok; Kumar, Nirmal; Chandra, Nagasuma R; Surolia, Avadhesha

    2008-04-01

    The role of lectins in mediating cancer metastasis, apoptosis as well as various other signaling events has been well established in the past few years. Data on various aspects of the role of lectins in cancer is being accumulated at a rapid pace. The data on lectins available in the literature is so diverse, that it becomes difficult and time-consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. Not only do the lectins vary significantly in their individual functional roles, but they are also diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities and specificities as well as their potential applications. An organization of these seemingly independent data into a common framework is essential in order to achieve effective use of all the data towards understanding the roles of different lectins in different aspects of cancer and any resulting applications. An integrated knowledge base (CancerLectinDB) together with appropriate analytical tools has therefore been developed for lectins relevant for any aspect of cancer, by collating and integrating diverse data. This database is unique in terms of providing sequence, structural, and functional annotations for lectins from all known sources in cancer and is expected to be a useful addition to the number of glycan related resources now available to the community. The database has been implemented using MySQL on a Linux platform and web-enabled using Perl-CGI and Java tools. Data for individual lectins pertain to taxonomic, biochemical, domain architecture, molecular sequence and structural details as well as carbohydrate specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value for various studies on lectin cancer biology. CancerLectinDB can be accessed through

  20. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  1. Refolding and characterization of the functional ligand-binding domain of human lectin-like oxidized LDL receptor.

    Science.gov (United States)

    Xie, Qiuhong; Matsunaga, Shigeru; Shi, Xiaohua; Ogawa, Setsuko; Niimi, Setsuko; Wen, Zhesheng; Tokuyasu, Ken; Machida, Sachiko

    2003-11-01

    Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a type II membrane protein that can recognize a variety of structurally unrelated macromolecules, plays an important role in host defense and is implicated in atherogenesis. To understand the interaction between human LOX-1 and its ligands, in this study the functional C-type lectin-like domain (CTLD) of LOX-1 was reconstituted at high efficiency from inactive aggregates in Escherichia coli using a refolding technique based on an artificial chaperone. The CD spectra of the purified domain suggested that the domain has alpha-helical structure and the blue shift of Trp residues was observed on refolding of the domain. Like wild-type hLOX-1, the refolded CTLD domain was able to bind modified LDL. Thus, even though CTLD contains six Cys residues that form disulfide bonds, it recovered its specific binding ability on refolding. This suggests that the correct disulfide bonds in CTLD were formed by the artificial chaperone technique. Although the domain lacked N-glycosylation, it showed high affinity for its ligand in surface plasmon resonance experiments. Thus, unglycosylated CTLD is sufficient for binding modified LDL.

  2. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    International Nuclear Information System (INIS)

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  3. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  4. Lectin-enzyme binding assays : development of the technique and applications in biochemistry and medicine

    NARCIS (Netherlands)

    J.M. Pekelharing

    1989-01-01

    textabstractThe aim of this work is to determine if lectins can be used in "sandwich" ELISA techniques so that the glycosylation of specific proteins in mixtures could be characterised in a fast and sensitive way without prior purification of the protein. Furthermore, the feasability of

  5. Interaction of lectins with membrane receptors on erythrocyte surfaces.

    Science.gov (United States)

    Sung, L A; Kabat, E A; Chien, S

    1985-08-01

    The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.

  6. Increased Autoreactivity of the Complement-Activating Molecule Mannan-Binding Lectin in a Type 1 Diabetes Model

    Directory of Open Access Journals (Sweden)

    Jakob Appel Østergaard

    2016-01-01

    Full Text Available Background. Diabetic kidney disease is the leading cause of end-stage renal failure despite intensive treatment of modifiable risk factors. Identification of new drug targets is therefore of paramount importance. The complement system is emerging as a potential new target. The lectin pathway of the complement system, initiated by the carbohydrate-recognition molecule mannan-binding lectin (MBL, is linked to poor kidney prognosis in diabetes. We hypothesized that MBL activates complement upon binding within the diabetic glomerulus. Methods. We investigated this by comparing complement deposition and activation in kidneys from streptozotocin-induced diabetic mice and healthy control mice. Results. After 20 weeks of diabetes, glomerular deposition of MBL was significantly increased. Diabetic animals had 2.0-fold higher (95% CI 1.6–2.5 immunofluorescence intensity from anti-MBL antibodies compared with controls (P<0.001. Diabetes and control groups did not differ in glomerular immunofluorescence intensity obtained by antibodies against complement factors C4, C3, and C9. However, the circulating complement activation product C3a was increased in diabetes as compared to control mice (P=0.04. Conclusion. 20 weeks of diabetes increased MBL autoreactivity in the kidney and circulating C3a concentration. Together with previous findings, these results indicate direct effects of MBL within the kidney in diabetes.

  7. Comparison of the antimicrobial adhesion potential of human body fluid glycoconjugates using fucose-binding lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus lectin (UEA-I).

    Science.gov (United States)

    Lerrer, Batia; Lesman-Movshovich, Efrat; Gilboa-Garber, Nechama

    2005-09-01

    Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are "secretors" express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of "secretors" and "nonsecretors" with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that "nonsecretor" body fluids were not less efficient than those of "secretors" in PA-IIL blocking. UEA-I, which interacted only with the "secretors" glycoproteins, was most sensitive to those of the seminal fluids.

  8. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  9. Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins

    DEFF Research Database (Denmark)

    Thiel, Steffen

    2007-01-01

    Mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin are all complement activating soluble pattern recognition molecules with recognition domains linked to collagen-like regions. All four may form complexes with four structurally related proteins, the three MBL-associated serine...... proteases (MASPs), MASP-1, MASP-2 and MASP-3, and a smaller MBL-associated protein (MAp19). The four recognition molecules recognize patterns of carbohydrate or acetyl-group containing ligands. After binding to the relevant targets all four are able to activate the complement system. We thus have a system...... where four different and/or overlapping patterns of microbial origin or patterns of altered-self may be recognized, but in all cases the signalling molecules, the MASPs, are shared. MASP-1 and MASP-3 are formed from one gene, MASP1/3, by alternative splicing generating two different mRNAs from a single...

  10. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  11. LIBRA: LIgand Binding site Recognition Application.

    Science.gov (United States)

    Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

    2015-12-15

    In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Lectin-Array Blotting.

    Science.gov (United States)

    Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

    2017-09-01

    Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  13. High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

    Science.gov (United States)

    2010-06-01

    host defense against a wide range of viral and other pathogens. MBL is a C-type lectin that recognizes hexose sugars including man- nose, glucose...should be evaluatedmore broadly as an immunotherapeutic agent for a wide spectrum of glycosylated pathogens. MATERIALS AND METHODS Production and... coagulation mod- ulators, antisense technologies, therapeutic antibodies and Table 1. Pharmacokinetic Parameters of Low- vs High-Dose Recombinant Human

  14. High levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis

    Directory of Open Access Journals (Sweden)

    K.A. Miranda

    2009-04-01

    Full Text Available The clinical heterogeneity observed in leptospirosis may be associated with host factors or bacteria virulence. Human serum mannose-binding lectin (MBL recognizes many pathogens, and low levels of this lectin are associated with susceptibility to infection. MBL is also implicated in the modulation of the inflammatory process. We determined the levels of serum MBL during leptospirosis infection. A double-antibody sandwich ELISA was used to detect the immunoreactive serum MBL. The ELISA plates were coated with monoclonal antibody to MBL and bound MBL or recombinant human MBL were detected by rabbit anti-human MBL serum. HRPO-conjugated goat anti-rabbit antibody was used for detection of the reaction. Two groups of patients seen at referral hospitals in Recife, PE, Brazil, were divided according to the year of infection, 2001 (N = 61 or 2002 (N = 57 and compared in terms of disease severity and levels of serum MBL. A group of healthy volunteers (N = 97 matched by age, gender, and ethnic background was used as control. Patients infected in 2001 had more severe outcomes than those infected in 2002, including jaundice, hemorrhage, respiratory alteration, and renal complication (P = 0.0009; chi-square test. The frequency of patients producing serum MBL >1000 ng/mL was higher in the 2001 group than in the 2002 and control groups (P < 0.01, suggesting an association of MBL level with disease severity. The involvement of MBL and genetic variation of the MBL2 gene should be further evaluated to establish the role of this lectin in the pathogenesis of leptospirosis.

  15. Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

    Science.gov (United States)

    Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

    2016-01-01

    Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses. Copyright © 2016 the American Physiological Society.

  16. The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+ cells

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2012-02-01

    Full Text Available It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4(+) cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit...

  17. Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B isolectin to alphagalactosyl carbohydrate antigens in the surface phase

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Winter, Harry C; Goldstein, Irwin J

    2004-01-01

    The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and lam...

  18. Influence of a yeast fermented product on the serum levels of the mannan-binding lectin and the antibodies against the Newcastle disease virus in Ross broilers

    DEFF Research Database (Denmark)

    Cortés-Coronado, R F; Gómez-Rosales, S; de L Angeles, M

    2017-01-01

    The objective of this research was to evaluate the serum concentrations of mannan-binding lectin (MBL) at different ages in Ross broilers fed increasing amounts of a yeast-fermented product (YFP) and inoculated with a vaccine against Newcastle disease virus (NDV). Eighty mixed Ross B308 broilers...

  19. Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

    DEFF Research Database (Denmark)

    Alves Pedroso, ML; Boldt, AB; Pereira-Ferrari, L

    2008-01-01

    Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2...

  20. Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis

    DEFF Research Database (Denmark)

    Skjødt, Mikkel-Ole; Palarasah, Yaseelan; Rasmussen, Karina Juhl

    2010-01-01

    The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intest...... protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway....

  1. Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies.

    Science.gov (United States)

    Singh, D D; Saikrishnan, K; Kumar, Prashant; Surolia, A; Sekar, K; Vijayan, M

    2005-10-01

    The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.

  2. Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruignosa

    DEFF Research Database (Denmark)

    Møller-Kristensen, Mette; Ip, WK; Shi, L

    2006-01-01

    studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5......Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical...... injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from...

  3. Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora damicornis.

    Science.gov (United States)

    Zhou, Zhi; Yu, Xiaopeng; Tang, Jia; Zhu, Yunjie; Chen, Guangmei; Guo, Liping; Huang, Bo

    2017-05-01

    Rhamnose-binding lectin (RBL) is a type of Ca 2+ -independent lectin with tandem repeat carbohydrate-recognition domain, and is crucial for the innate immunity in many invertebrates. In this study, the cDNA sequence encoding RBL in coral Pocillopora damicornis (PdRBL-1) was cloned. The PdRBL-1 protein shared highest amino acid sequence similarity (55%) with the polyp of Hydra vulgaris, and contained a signal peptide and two tandem carbohydrate-recognition domains in which all cysteine residues were conserved. Surface plasmon resonance method revealed that the recombinant PdRBL-1 protein bound to LPS and Lipid A, but not to LTA, β-glucan, mannose and Poly (I:C). Results also showed that it bonded with zooxanthellae using western blotting method, and that the bound protein was detectable only at concentrations higher than 10 2 zooxanthellae cell mL -1 . When recombinant PdRBL-1 protein was preincubated with LPS, lower amounts of protein bound to zooxanthellae compared to cells not preincubated with LPS. Furthermore, PdRBL-1 mRNA expression increased significantly at 12 h, and declined to the baseline at 24 h after heat stress at 31 °C. These results collectively suggest that PdRBL-1 could recognize not only pathogenic bacteria but also symbiotic zooxanthellae, and that the recognition of zooxanthellae by PdRBL-1 could be repressed by pathogenic bacteria through competitive binding. This information allows us to gain new insights in the mechanisms influencing the establishment and maintenance of coral-zooxanthella symbiosis in coral P. damicornis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Adenanthera pavonina L. galactomannan: application for isolation galactose-binding lectins

    International Nuclear Information System (INIS)

    Tavares, Ricardo O.; Moreira, Renato A.

    2001-01-01

    The Adenanthera pavonina L. (Carolina) endospermic gum was investigated in relation to minimal composition and to its ability in purifying lectins. These, proteins specifically interact with cell surface carbohydrates, being better isolated by affinity chromatography, in a matrix containing these oligosaccharides. Carolina seed gum is hydrophilic, and as other known endospermic gums, is a classic galactomannan, constituted by a repeat structure of D-man p units connected by a β-(1→4) linkage, with D-gal p units connected by a α-(1→6) linkage in the ramifications. The ratio M:G determined by 13 C-nuclear magnetic resonance spectroscopy was 1,8:1. Affinity chromatography were realized in Carolina gum columns, treated with epichlorhydrin 1, 2, 3 and 4 M. Affinity columns, prepared with these polysaccharides, were tested for the purification of various galactose-specific lectin. The epichlorhydrin 3M treatment was compared with that suggested by APPUKUTTAN et al. (1977), and in some cases, the behavior was quite different, probably due to differences between the studied gums. (author)

  5. Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or SAP trigger cross-activation of the complement system

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Doni, Andrea; Skjødt, Mikkel-Ole

    2011-01-01

    The long pentraxin 3 (PTX3), serum amyloid P component (SAP) and C-reactive protein (CRP) belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via...... the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain, but not CRP. MBL:PTX3 complex formation resulted in recruitment of C......1q, but this was not seen for the MBL:SAP complex. However, both MBL:PTX3 and MBL:SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 lead to communication between the lectin and classical complement...

  6. Lectindb: a plant lectin database.

    Science.gov (United States)

    Chandra, Nagasuma R; Kumar, Nirmal; Jeyakani, Justin; Singh, Desh Deepak; Gowda, Sharan B; Prathima, M N

    2006-10-01

    Lectins, a class of carbohydrate-binding proteins, are now widely recognized to play a range of crucial roles in many cell-cell recognition events triggering several important cellular processes. They encompass different members that are diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities, and specificities as well as their larger biological roles and potential applications. It is not surprising, therefore, that the vast amount of experimental data on lectins available in the literature is so diverse, that it becomes difficult and time consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. To achieve an effective use of all the data toward understanding the function and their possible applications, an organization of these seemingly independent data into a common framework is essential. An integrated knowledge base ( Lectindb, http://nscdb.bic.physics.iisc.ernet.in ) together with appropriate analytical tools has therefore been developed initially for plant lectins by collating and integrating diverse data. The database has been implemented using MySQL on a Linux platform and web-enabled using PERL-CGI and Java tools. Data for each lectin pertain to taxonomic, biochemical, domain architecture, molecular sequence, and structural details as well as carbohydrate and hence blood group specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value not only for basic studies in lectin biology but also for basic studies in pursuing several applications in biotechnology, immunology, and clinical practice, using these molecules.

  7. F-Type Lectins: A Highly Diversified Family of Fucose-Binding Proteins with a Unique Sequence Motif and Structural Fold, Involved in Self/Non-Self-Recognition

    Directory of Open Access Journals (Sweden)

    Gerardo R. Vasta

    2017-11-01

    Full Text Available The F-type lectin (FTL family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD] that displays a novel jellyroll fold (“F-type” fold and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms that are expressed in a single individual has revealed genetic mechanisms of diversification in ligand recognition that are unique to FTLs. Functions of FTLs in self/non-self-recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others. In addition, although the F-type fold is distinctive for FTLs, a structure-based search revealed apparently unrelated proteins with minor sequence similarity to FTLs that displayed the FTLD fold. In general, the phylogenetic analysis of FTLD sequences from viruses to mammals reveals clades that are consistent with the currently accepted taxonomy of extant species. However, the surprisingly discontinuous distribution of FTLDs within each taxonomic category suggests not only an extensive structural/functional diversification of the FTLs along evolutionary lineages but also that this intriguing lectin family has been subject to frequent gene duplication, secondary loss, lateral transfer, and functional co-option.

  8. Adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations

    DEFF Research Database (Denmark)

    Kjærup, Rikke Munkholm; Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann

    2014-01-01

    in the pathogenesis of IBV infection and the production of IBV-specific antibodies, which may be exploited in optimising IBV vaccine strategies. The present study shows that MBL has the capability to bind to IBV in vitro. Chickens from two inbred lines (L10H and L10L) selected for high or low MBL serum concentrations......, respectively, were vaccinated against IBV with or without the addition of the MBL ligands mannan, chitosan and fructooligosaccharide (FOS). The addition of MBL ligands to the IBV vaccine, especially FOS, enhanced the production of IBV-specific IgG antibody production in L10H chickens, but not L10L chickens...... to the vaccine, most pronouncedly after the first vaccination. As MBL ligands co-administered with IBV vaccine induced differences between the two chicken lines, these results indirectly suggest that MBL is involved in the immune response to IBV vaccination. Furthermore, the higher antibody response in L10H...

  9. Analysis of myelomonocytic leukemic differentiation by a cell surface marker panel including a fucose-binding lectin from Lotus tetragonolobus.

    Science.gov (United States)

    Elias, L; Van Epps, D E

    1984-06-01

    The fucose-binding lectin from Lotus tetragonolobus ( FBL -L) has been previously shown to bind specifically to normal cells of the myeloid and monocytic lineages. The purpose of this study was to explore the utility of fluoresceinated FBL -L as a leukemia differentiation marker in conjunction with a panel of other frequently used surface markers (Fc receptor, HLA-DR, OKM1, and antimonocyte antibody). FBL -L reacted with leukemic cells in 8/9 cases of clinically recognized acute myeloid leukemia, including myeloid blast crisis of chronic granulocytic leukemia, 3/3 cases of chronic phase chronic myelogenous leukemia, and in 2/7 cases of clinically undifferentiated acute leukemia. Correlations were noted between reactivity with FBL -L, and DR and Fc receptor expression. Among continuous cell lines, FBL -L bound with high intensity to a majority of HL-60 and U937 cells. The less well differentiated myeloblast cell lines, KG-1, KG1a , and HL-60 blast II, exhibited less FBL -L binding than HL-60 and U937. A moderate proportion of K562 cells exhibited low level binding of FBL -L. Several lymphoblastic cell lines exhibited a pattern of low intensity binding that was distinguishable from the high intensity binding pattern of the myeloblastic lines. FBL -L reactivity of U937 was enhanced by induction of differentiation with leukocyte conditioned medium, but not dimethylsulfoxide. Such treatments induced contrasting patterns of change of HL-60 and U937 when labeled with OKM1, alpha-Mono, and HLA-DR. These studies demonstrate the application of FBL -L to analysis and quantitation of myelomonocytic leukemic differentiation.

  10. A novel C-type lectin from the sea cucumber Apostichopus japonicus (AjCTL-2) with preferential binding of d-galactose.

    Science.gov (United States)

    Wang, Hui; Xue, Zhuang; Liu, Zhaoqun; Wang, Weilin; Wang, Feifei; Wang, Ying; Wang, Lingling; Song, Linsheng

    2018-05-15

    C-type lectins (CTLs) are Ca 2+ dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains (CRDs). In the present study, a novel CTL was identified from sea cucumber Apostichopus japonicus (named as AjCTL-2). The deduced amino acid sequence of AjCTL-2 was homologous to CTLs from other animals with the identities ranging from 33% to 40%. It contained a canonical signal peptide at the N-terminus, a low density lipoprotein receptor class A (LDLa), a C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), and a CRD with two motifs Glu-Pro-Asn (EPN) and Trp-Asn-Asp (WND) in Ca 2+ binding site 2. The mRNA transcripts of AjCTL-2 were extensively expressed in all the tested tissues including respiratory tree, muscle, gut, coelomocyte, tube-foot, body wall and gonad, and the highest expression level of AjCTL-2 in coelomocyte was about 4.2-fold (p sea cucumber. Copyright © 2018. Published by Elsevier Ltd.

  11. Role of aromatic amino acids in carbohydrate binding of plant lectins : Laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins

    NARCIS (Netherlands)

    Siebert, HC; vonderLieth, CW; Kaptein, R; Beintema, JJ; Dijkstra, K; vanNuland, N; Soedjanaatmadja, UMS; Rice, A; Vliegenthart, JFG; Wright, CS; Gabius, HJ

    Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye.

  12. Extreme high prevalence of a defective mannose-binding lectin (MBL2 genotype in native South American West Andean populations.

    Directory of Open Access Journals (Sweden)

    José Raul Sandoval

    Full Text Available Mannose-binding lectin (MBL is one of the five recognition molecules in the lectin complement pathway. Common variant alleles in the promoter and structural regions of the human MBL gene (MBL2 influence the stability and serum concentration of the protein. Epidemiological studies have shown that MBL2 variant alleles are associated with susceptibility to and the course of different types of infectious and inflammatory conditions. However, it has been suggested that these alleles are maintained in different populations due to selected advantages for carriers. We investigated the MBL2 allelic variation in indigenous individuals from 12 different West Central South America localities spanning from the desert coast, high altitude Andean plates and the Amazon tropical forest within the territories of Peru (n = 249 (Departments of Loreto, Ucayali, Lambayeque, Junin, Ayacucho, Huancayo and Puno, and Ecuador (n = 182 (Region of Esmeraldas and Santo Domingo de los Colorados. The distribution of MBL2 genotypes among the populations showed that the defective variant LYPB haplotype was very common. It showed the highest frequencies in Puno (Taquile (0.80, Amantani (0.80 and Anapia (0.58 islander communities of the Lake Titicaca, but lower frequencies of 0.22 in Junin (Central Andean highland and Ucayali (Central Amazonian forest, as well as 0.27 and 0.24 in the Congoma and Cayapa/Chachis populations in the Amazonian forest in Ecuador were also observed. Our results suggest that the high prevalence of the MBL2 LYPB variant causing low levels of functional MBL in serum may mainly reflect a random distribution due to a population bottleneck in the founder populations.

  13. Extreme high prevalence of a defective mannose-binding lectin (MBL2) genotype in native South American West Andean populations.

    Science.gov (United States)

    Sandoval, José Raul; Madsen, Hans O; De Stefano, Gianfranco; Descailleaux-Dulanto, Jaime; Velazquez-Reinoso, Margarita; Ñique, Cesar; Fujita, Ricardo; Garred, Peter

    2014-01-01

    Mannose-binding lectin (MBL) is one of the five recognition molecules in the lectin complement pathway. Common variant alleles in the promoter and structural regions of the human MBL gene (MBL2) influence the stability and serum concentration of the protein. Epidemiological studies have shown that MBL2 variant alleles are associated with susceptibility to and the course of different types of infectious and inflammatory conditions. However, it has been suggested that these alleles are maintained in different populations due to selected advantages for carriers. We investigated the MBL2 allelic variation in indigenous individuals from 12 different West Central South America localities spanning from the desert coast, high altitude Andean plates and the Amazon tropical forest within the territories of Peru (n = 249) (Departments of Loreto, Ucayali, Lambayeque, Junin, Ayacucho, Huancayo and Puno), and Ecuador (n = 182) (Region of Esmeraldas and Santo Domingo de los Colorados). The distribution of MBL2 genotypes among the populations showed that the defective variant LYPB haplotype was very common. It showed the highest frequencies in Puno (Taquile (0.80), Amantani (0.80) and Anapia (0.58) islander communities of the Lake Titicaca), but lower frequencies of 0.22 in Junin (Central Andean highland) and Ucayali (Central Amazonian forest), as well as 0.27 and 0.24 in the Congoma and Cayapa/Chachis populations in the Amazonian forest in Ecuador were also observed. Our results suggest that the high prevalence of the MBL2 LYPB variant causing low levels of functional MBL in serum may mainly reflect a random distribution due to a population bottleneck in the founder populations.

  14. [Effect of thyroid hormones on the histotopography of lectin receptors in the rat salivary gland].

    Science.gov (United States)

    Lutsik, A D; Iashchenko, A M; Detiuk, E S

    1987-04-01

    Using lectin-peroxidase technique, the influence of hypo- and hyperthyroidism on histotopography of glycoconjugates has been investigated in rat submandibular gland. The following lectins were used: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Laburnum anagyroides lectin (LAL) and concanavalin A (con A). It has been demonstrated that hyperthyroidism is accompanied by the loss of con A, WGA and LAL receptor sites. Hypothyrodism enhanced con A binding to granular duct cells with a parallel reduction in WGA and LAL binding to these or other duct cells. Hypothyroidism as well as hyperthyroidism markedly enhanced PNA binding to duct epitheliocytes with redistribution of these lectin binding sites from the luminal surface of salivary ducts into the cytoplasm of duct cells. Possible interpretations of the observed phenomena are discussed.

  15. Lectins from Mycelia of Basidiomycetes

    Directory of Open Access Journals (Sweden)

    Valentina E. Nikitina

    2017-06-01

    Full Text Available Lectins are proteins of a nonimmunoglobulin nature that are capable of specific recognition of and reversible binding to the carbohydrate moieties of complex carbohydrates, without altering the covalent structure of any of the recognized glycosyl ligands. They have a broad range of biological activities important for the functioning of the cell and the whole organism and, owing to the high specificity of reversible binding to carbohydrates, are valuable tools used widely in biology and medicine. Lectins can be produced by many living organisms, including basidiomycetes. Whereas lectins from the fruit bodies of basidiomycetes have been studied sufficiently well, mycelial lectins remain relatively unexplored. Here, we review and comparatively analyze what is currently known about lectins isolated from the vegetative mycelium of macrobasidiomycetes, including their localization, properties, and carbohydrate specificities. Particular attention is given to the physiological role of mycelial lectins in fungal growth and development.

  16. Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Kang, Hee Jung; Kim, Ji Yeon

    2013-01-01

    It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...... cytokine production by LPS-mediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells.......It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...

  17. Serum levels of chicken mannan-binding lectin (MBL) during virus infections; indication that chicken MBL is an acute phase reactant

    DEFF Research Database (Denmark)

    Nielsen, O.L.; Jensenius, J. C.; Jørgensen, Poul Henrik

    1999-01-01

    Mannan-binding lectin (MBL) is a serum collectin which is believed to be an opsonin of the innate immune defence against various microorganisms. MBL is a minor acute phase reactant in man. We investigated the concentration of serum MBL in chickens infected with infectious bronchitis virus (IBV...... levels returned to normal values 6-10 days after infection. The results indicated that MBL is a minor acute phase reactant in chickens....

  18. Genetically determined high serum levels of mannose-binding lectin and agalactosyl IgG are associated with ischemic heart disease in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Troelsen, Lone N; Garred, Peter; Madsen, Hans O.

    2007-01-01

    Patients with rheumatoid arthritis (RA) have excess morbidity and mortality due to ischemic heart disease. It has been suggested that high serum levels of mannose-binding lectin (MBL) and agalactosyl IgG (IgG-G0) are associated with increased inflammation in RA. MBL also enhances inflammation......-mediated tissue injury during postischemic reperfusion. This study was undertaken to examine whether these factors are associated with increased risk of ischemic heart disease in RA....

  19. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    Science.gov (United States)

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Vitelline coat of Unio elongatulus: III. Glycan chain analysis of the 220- and 180-kD components by means of lectins.

    Science.gov (United States)

    Focarelli, R; Leotta, F; Lampariello, R; Rosati, F

    1995-02-01

    Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220- and 180-kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA1 reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N-linked high mannose type oligosaccharide chains, bound specifically to the 180-kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O-linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220-kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220-kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180-kD protein is part of the entire vc structure, the 220-kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220-kD protein acts as a ligand molecule in the sperm-egg interaction.

  1. The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

    Science.gov (United States)

    Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

    2000-04-01

    Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

  2. Polymorphisms in Genes Coding for Cytokines, Mannose-Binding Lectin, Collagen Metabolism and Thrombophilia in Women with Cervical Insufficiency

    DEFF Research Database (Denmark)

    Sundtoft, Iben; Uldbjerg, Niels; Steffensen, Rudi

    2015-01-01

    OBJECTIVE: To study the association between cervical insufficiency and single nucleotide polymorphisms in seven genes coding for pro- and anti-inflammatory cytokine-related factors, mannose-binding lectin 2 (MBL2), collagen1α1 (COL1A1), factor II and factor V Leiden genes. METHODS: In a case......-control study, potential maternal biomarkers for cervical insufficiency were investigated in 30 women with a history of second-trimester miscarriage or preterm birth due to cervical insufficiency and in 70 control women. RESULTS: Homozygous carriers of the interleukin 6 (IL6) -174 genotype GG had an odds ratio...... (OR) of 3.1 [95% confidence interval (95% CI) 1.3-7.4, p = 0.01] and MBL2 genotypes coding for low or intermediate levels of plasma MBL had an OR of 3.3 (95% CI 1.2-9.0, p = 0.01) for cervical insufficiency compared with controls. Serum MBL levels were lower in women with cervical insufficiency than...

  3. Glycoprotein profiles of macrophages at different stages of activation as revealed by lectin binding after electrophoretic separation.

    Science.gov (United States)

    Irimura, T; North, S M; Nicolson, G L

    1987-01-01

    Glycoprotein profiles of rat macrophages (M phi) at different stages of activation were studied by examining the reactivity of various lectins to the glycoproteins separated by polyacrylamide gel electrophoresis. Ricinus communis agglutinin 1 (RCA1) revealed several components including glycoproteins of Mr 160 kDa and 65 kDa prominent in resident M phi. A pokeweed mitogen (PWM) isolectin, Pa-4, recognizes branched poly(N-acetyllactosamine)-type carbohydrate chains, and revealed a significant increase in glycoproteins of Mr ranging from 70 kDa to 150 kDa on thioglycolate-elicited M phi. Increased reactivity of PWM to thioglycolate-elicited M phi was observed by direct binding of 125I-labeled Pa-4 to intact or glutaraldehyde-fixed M phi. Histochemical staining of formaldehyde-fixed M phi in vitro with biotinylated Pa-4 was consistent with the gel analysis, that is, resident M phi had no reactivity while thioglycolate-elicited M phi showed slight reactivity. Alveolar and intratumoral M phi bound more Pa-4 than resident or thioglycolate-elicited M phi. The PWM isolectin may therefore serve as a marker for an early stage of M phi activation.

  4. The lectin from Musa paradisiaca binds with the capsid protein of tobacco mosaic virus and prevents viral infection.

    Science.gov (United States)

    Liu, Xiao-Yu; Li, Huan; Zhang, Wei

    2014-05-04

    It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro , and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo , purified rBanLec-1 exhibited more significant effects on TMV infection in vitro . Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.

  5. Circulating levels of mannan-binding lectin (MBL) and MBL-associated serine protease 2 in endemic pemphigus foliaceus

    DEFF Research Database (Denmark)

    Messias-Reason, I; Bosco, DG; Nisihara, RM

    2008-01-01

    Endemic pemphigus foliaceus (EPF) is an autoimmune disease, which occurs in Brazil and other regions of South America. Mannose-binding lectin (MBL) and MBL-associated serine protease (MASP-2) play a key role in innate immunity, and its deficiency has been related to increased susceptibility...... to infection and autoimmune diseases. MBL and MASP-2 serum levels were measured in 114 patients with EPF and in 100 healthy individuals in Brazil. MBL and MASP-2 levels were measured by sandwich assays (time-resolved immunofluorimetic assay) using monoclonal antibodies. No difference was observed in the MBL...... level in patients with EPF compared with controls [mean +/- SEM 1230.07 +/- 132.18 ng/mL (median 789.0 ng/mL) vs. 1036.98 +/- 117.99 ng/mL (median 559.5 ng/mL), P = 0.32]. Non-significant lower MASP-2 levels were observed in EPF [274.34 +/- 15.66 ng/mL (median 239.5 ng/mL ) vs. 304.72 +/- 15.28 ng...

  6. Genotyping of mannose-binding lectin (MBL2) codon 54 and ...

    African Journals Online (AJOL)

    Rabah M. Shawky

    2013-11-17

    Nov 17, 2013 ... ing attachment, ingestion and killing of opsonized pathogens by phagocytes [5] ..... pneumonia and culture-proven sepsis during the first month of life [26]. ..... variation in the serum concentration of mannan-binding protein. Acta Paediatr ... phase reactant in adults with community-acquired pneumococcal.

  7. An assay for the mannan-binding lectin pathway of complement activation

    DEFF Research Database (Denmark)

    Petersen, Steen Vang; Thiel, S; Jensen, L

    2001-01-01

    activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt...

  8. Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

    Science.gov (United States)

    Poiroux, Guillaume; Barre, Annick; van Damme, Els J. M.; Benoist, Hervé; Rougé, Pierre

    2017-01-01

    Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. PMID:28598369

  9. Mannose-binding lectin and Ficolin-2 gene polymorphisms predispose to cytomegalovirus (re)infection after orthotopic liver transplantation

    NARCIS (Netherlands)

    de Rooij, Bert-Jan F.; van der Beek, Martha T.; van Hoek, Bart; Vossen, Ann C. T. M.; ten Hove, W. Rogier; Roos, Anja; Schaapherder, Alexander F.; Porte, Robert J.; van der Reijden, Johan J.; Coenraad, Minneke J.; Hommes, Daniel W.; Verspaget, Hein W.

    2011-01-01

    Background & Aims: The lectin pathway of complement activation is a crucial effector cascade of the innate immune response to pathogens. Cytomegalovirus (CMV) infection occurs frequently in immunocompromised patients after orthotopic liver transplantation (OLT). Single-nucleotide polymorphisms

  10. Lectins and their application to clinical microbiology.

    OpenAIRE

    Slifkin, M; Doyle, R J

    1990-01-01

    Lectins are generally associated with plant or animal components, selectively bind carbohydrates, and interact with procaryotic and eucaryotic cells. Lectins have various specificities that are associated with their ability to interact with acetylaminocarbohydrates, aminocarbohydrates, sialic acids, hexoses, pentoses, and as other carbohydrates. Microbial surfaces generally contain many of the sugar residues that react with lectins. Lectins are presently used in the clinical laboratory to typ...

  11. Two factors of the lectin pathway of complement, l-ficolin and mannan-binding lectin, and their associations with prematurity, low birthweight and infections in a large cohort of Polish neonates.

    Science.gov (United States)

    Swierzko, Anna St; Atkinson, Anne P M; Cedzynski, Maciej; Macdonald, Shirley L; Szala, Agnieszka; Domzalska-Popadiuk, Iwona; Borkowska-Klos, Monika; Jopek, Aleksandra; Szczapa, Jerzy; Matsushita, Misao; Szemraj, Janusz; Turner, Marc L; Kilpatrick, David C

    2009-02-01

    Ficolins and one collectin, mannan-binding lectin (MBL), are the only factors known to activate the lectin pathway (LP) of complement. There is considerable circumstantial evidence that MBL insufficiency can increase susceptibility to various infections and influence the course of several non-infectious diseases complicated by infections. Much less information is available concerning l-ficolin. We report the results of a prospective study to investigate any association between either MBL deficiency or l-ficolin deficiency with prematurity, low birthweight or perinatal infections in a large cohort of Polish neonates, representing an ethnically homogenous population (n=1832). Cord blood samples were analysed to determine mbl-2 gene variants, MBL concentrations and MBL-MASP-2 complex activities (MBL-dependent lectin pathway activity) as well as l-ficolin levels. Median concentrations of l-ficolin and MBL were 2500 and 1124 ng/ml, respectively, while median LP activity was 272 mU/ml. After genotyping, 60.6% of babies were mbl-2 A/A, 35.4% were A/O and 4% were O/O genotypes. We found relative l-ficolin deficiency to be associated with prematurity, low birthweight and infections. l-Ficolin concentration correlated with gestational age and with birthweight, independently of gestational age. Preterm deliveries (prematurity. Our findings suggest that l-ficolin participates in host defence during the perinatal period and constitute the first evidence that relative l-ficolin deficiency may contribute to the adverse consequences of prematurity. Some similar trends were found with facets of MBL deficiency, but the observed relationships were weaker and less consistent.

  12. Mannose-binding lectin and l-ficolin polymorphisms in patients with community-acquired pneumonia caused by intracellular pathogens.

    Science.gov (United States)

    van Kempen, Gijs; Meijvis, Sabine; Endeman, Henrik; Vlaminckx, Bart; Meek, Bob; de Jong, Ben; Rijkers, Ger; Bos, Willem Jan

    2017-05-01

    Community-acquired pneumonia (CAP) is the leading infectious disease requiring hospitalization in the western world. Genetic variability affecting the host response to infection may play a role in susceptibility and outcome in patients with CAP. Mannose-binding lectin (MBL) and l-ficolin (l-FCN) are two important activators of the complement system and they can enhance phagocytosis by opsonization. In a prospective cohort of 505 Dutch patients with CAP and 227 control participants we studied whether polymorphisms in the MBL (MBL2) and FCN (FCN2) genes influenced susceptibility and outcome. No difference in frequency of these genotypes was found between patients with CAP in general and controls. However, the +6424G>T single nucleotide polymorphism (SNP) in FCN2 was more common in patients with a Coxiella burnetii pneumonia (P = 0·014). Moreover, the haplotypes coding for the highest MBL serum levels (YA/YA and YA/XA) predisposed to atypical pneumonia (C. burnetii, Legionella or Chlamydia species or Mycoplasma pneumoniae) compared with controls (P = 0·016). Furthermore, patients with these haplotypes were more often bacteraemic (P = 0·019). It can therefore be concluded that MBL2 and FCN2 polymorphisms are not major risk factors for CAP in general, but that the +6424G>T SNP in the FCN2 gene predisposes to C. burnetii pneumonia. In addition, patients with genotypes corresponding with high serum MBL levels are at risk for atypical pneumonia, possibly caused by enhanced phagocytosis, thereby promoting cell entry of these intracellular bacteria. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

  13. Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

    International Nuclear Information System (INIS)

    Hiruma, Yoshiharu; Hirai, Takehiro; Tsuda, Eisuke

    2011-01-01

    Highlights: → Siglec-15 was identified as a gene overexpressed in giant cell tumor. → Siglec-15 mRNA expression increased in association with osteoclast differentiation. → Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D 3 and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

  14. Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Hiruma, Yoshiharu, E-mail: hiruma.yoshiharu.hy@daiichisankyo.co.jp [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Hirai, Takehiro [Translational Medicine and Clinical Pharmacology Department, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Tsuda, Eisuke [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan)

    2011-06-10

    Highlights: {yields} Siglec-15 was identified as a gene overexpressed in giant cell tumor. {yields} Siglec-15 mRNA expression increased in association with osteoclast differentiation. {yields} Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor {kappa}B ligand (RANKL) or tumor necrosis factor (TNF)-{alpha}. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D{sub 3} and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

  15. Development of gastrointestinal surface. VIII. Lectin identification of carbohydrate differences

    International Nuclear Information System (INIS)

    Pang, K.Y.; Bresson, J.L.; Walker, W.A.

    1987-01-01

    Binding of microvillus membranes (MVM) from newborn and adult rats by concanavalin A (Con A), Ulex europaeus (UEA I), Dolichos bifluorus (DBA), and Triticum vulgaris (WGA) was examined to determine the availability of carbohydrate-containing sites for these lectins on the intestinal surface during development. Consistent patterns of differences in the reaction of MVM with these lectins were found. Con A and UEA had much higher reactivities to MVM of adult than newborn rats. 125 I-labeled-UEA gel overlay experiments revealed the abundance of UEA-binding sites in MVM of adult rat in contrast to the two binding sites in MVM of a newborn rat. DBA bound only to MVM of the adults, and very few binding sites were found in immature MVM. In contrast to these lectins, WGA binding was much higher in MVM of the newborns and decreased with maturation. Additional experiments on the age dependence of UEA and DBA reactivities revealed that the most striking changes occur in animals from 2 to 2 wk of age. In MVM from 2-wk-old rats, there were only 13.9% and < 0.2% of the adult binding capacities for UEA and DBA, respectively. By the time the animals were 4 wk old, the binding capacity for UEA had attained close to the level of the adults, whereas for DBA it reached 71.3% of the adult value. These results provide definite evidence of changes in the intestinal surface during perinatal development

  16. Development of gastrointestinal surface. VIII. Lectin identification of carbohydrate differences

    Energy Technology Data Exchange (ETDEWEB)

    Pang, K.Y.; Bresson, J.L.; Walker, W.A.

    1987-05-01

    Binding of microvillus membranes (MVM) from newborn and adult rats by concanavalin A (Con A), Ulex europaeus (UEA I), Dolichos bifluorus (DBA), and Triticum vulgaris (WGA) was examined to determine the availability of carbohydrate-containing sites for these lectins on the intestinal surface during development. Consistent patterns of differences in the reaction of MVM with these lectins were found. Con A and UEA had much higher reactivities to MVM of adult than newborn rats. /sup 125/I-labeled-UEA gel overlay experiments revealed the abundance of UEA-binding sites in MVM of adult rat in contrast to the two binding sites in MVM of a newborn rat. DBA bound only to MVM of the adults, and very few binding sites were found in immature MVM. In contrast to these lectins, WGA binding was much higher in MVM of the newborns and decreased with maturation. Additional experiments on the age dependence of UEA and DBA reactivities revealed that the most striking changes occur in animals from 2 to 2 wk of age. In MVM from 2-wk-old rats, there were only 13.9% and < 0.2% of the adult binding capacities for UEA and DBA, respectively. By the time the animals were 4 wk old, the binding capacity for UEA had attained close to the level of the adults, whereas for DBA it reached 71.3% of the adult value. These results provide definite evidence of changes in the intestinal surface during perinatal development.

  17. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  18. Isolation and characterization of rhamnose-binding lectins from eggs of steelhead trout (Oncorhynchus mykiss) homologous to low density lipoprotein receptor superfamily.

    Science.gov (United States)

    Tateno, H; Saneyoshi, A; Ogawa, T; Muramoto, K; Kamiya, H; Saneyoshi, M

    1998-07-24

    Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 microg/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was L-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or Staphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) of D-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor

  19. Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

    Science.gov (United States)

    Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

    2012-01-01

    Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

  20. RNA sequencing based analysis of the spleen transcriptome following the infectious bronchitis virus infection of chickens selected for different mannose-binding lectin serum concentrations

    DEFF Research Database (Denmark)

    Hamzic, Edin; Kjærup, Rikke Brødsgaard; Mach, Núria

    2016-01-01

    in strategies to control IB. To this end, two chicken lines, selected for high and low serum concentration of mannose-binding lectin (MBL), a soluble pattern recognition receptor, were studied. In total, 32 animals from each line (designated L10H for high and L10L for low MBL serum concentration) were used....... Sixteen birds from each line were infected with IBV on day 1 and birds were euthanized at 1 week and 3 weeks post infection, 8 uninfected controls and 8 infected birds from each line at each occasion. RNA sequencing was performed on spleen samples from all 64 birds used in the experiment. Differential...

  1. Crystal Structure and Functional Characterization of the Complement Regulator Mannose-binding Lectin (MBL)/Ficolin-associated Protein-1 (MAP-1)

    DEFF Research Database (Denmark)

    Skjoedt, M.-o.; Roversi, P.; Hummelshoj, T.

    2012-01-01

    .5 nM, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homo-dimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer approximately 146 Angstrom...... long. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose dependent inhibitory effect on the lectin pathway activation, as measured by levels...

  2. Mannose-Binding Lectin Gene, MBL2, Polymorphisms Do Not Increase Susceptibility to Invasive Meningococcal Disease in a Population of Danish Children

    DEFF Research Database (Denmark)

    Lundbo, Lene F; Sørensen, Henrik T.; Clausen, Louise Nygaard

    2015-01-01

    of the innate immune system may predispose to invasive meningococcal disease (IMD). In this study, we investigated the effect of genetic variation in the mannose-binding lectin gene, MBL2, and its promoter on susceptibility to IMD and IMD-associated mortality among children. Methods.  Children (...Background.  Neisseria meningitidis is the cause of meningococcal bacteremia and meningitis, and nasopharyngeal colonization with this pathogen is common. The incidence of invasive disease is highest in infants, whereas adolescents more often are carriers. Altered regulation or dysfunction...

  3. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

    DEFF Research Database (Denmark)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig

    2007-01-01

    Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificit...

  4. The lectin pathway of complement

    DEFF Research Database (Denmark)

    Ballegaard, Vibe Cecilie Diederich; Haugaard, Anna Karen; Garred, P

    2014-01-01

    The pattern recognition molecules of the lectin complement pathway are important components of the innate immune system with known functions in host-virus interactions. This paper summarizes current knowledge of how these intriguing molecules, including mannose-binding lectin (MBL), Ficolin-1, -2......-1, -2 and -3 and CL-11 could have similar functions in HIV infection as the ficolins have been shown to play a role in other viral infections, and CL-11 resembles MBL and the ficolins in structure and binding capacity.......The pattern recognition molecules of the lectin complement pathway are important components of the innate immune system with known functions in host-virus interactions. This paper summarizes current knowledge of how these intriguing molecules, including mannose-binding lectin (MBL), Ficolin-1, -2...

  5. Autoradiographic localization of benzomorphan binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Crain, B.J.; Kwenjen Chang; McNamara, J.O.; Valdes, F.

    1985-07-17

    The benzomorphan subpopulation of opiate binding sites was labeled by (TH)diprenorphine in the presence of unlabeled ligands selected to quench and delta opiate binding sites. The distribution of benzomorphan binding sites was then localized autoradiographically. The distribution differs from the distributions of , delta and kappa opiate binding and is quite similar to the distribution of US -endorphin immunoreactivity. These observations support the hypothesis, based on biochemical studies in brain membranes, that benzomorphan binding sites may represent the ligand recognition sites of putative epsilon receptors. (Auth.). 34 refs.; 3 figs.

  6. Biological role of lectins: A review

    Directory of Open Access Journals (Sweden)

    K Kiran Kumar

    2012-01-01

    Full Text Available Lectins comprise a stracturally vary diverse class of proteins charecterized by their ability to selectively bind carbohydrate moieties of the glycoproteins of the cell surface. Lectins may be derived from plants, microbial or animal sources and may be soluble or membrane bound. Lectins is a tetramer made up of four nearly identical subunits. In human, lectins have been reported to cause food poisoning, hemolytic anemia, jaundice, digestive distress, protein and carbohydrate malabsorption and type I allergies. The present review focuses on the classification, structures, biological significance and application of lectins.

  7. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  8. Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

    Science.gov (United States)

    Hatakeyama, Tomomitsu; Ichise, Ayaka; Unno, Hideaki; Goda, Shuichiro; Oda, Tatsuya; Tateno, Hiroaki; Hirabayashi, Jun; Sakai, Hitomi; Nakagawa, Hideyuki

    2017-08-01

    The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure. © 2017 The Protein Society.

  9. A Lactose-Binding Lectin from the Marine Sponge Cinachyrella Apion (Cal) Induces Cell Death in Human Cervical Adenocarcinoma Cells

    Science.gov (United States)

    Rabelo, Luciana; Monteiro, Norberto; Serquiz, Raphael; Santos, Paula; Oliveira, Ruth; Oliveira, Adeliana; Rocha, Hugo; Morais, Ana Heloneida; Uchoa, Adriana; Santos, Elizeu

    2012-01-01

    Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5–10 µg/mL). Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 (10 µg/mL) for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer. PMID:22690140

  10. A Lactose-Binding Lectin from the Marine Sponge Cinachyrella Apion (Cal Induces Cell Death in Human Cervical Adenocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Adriana Uchoa

    2012-03-01

    Full Text Available Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5–10 µg/mL. Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 (10 µg/mL for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer.

  11. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites.

    Science.gov (United States)

    Friedrich, Nikolas; Santos, Joana M; Liu, Yan; Palma, Angelina S; Leon, Ester; Saouros, Savvas; Kiso, Makoto; Blackman, Michael J; Matthews, Stephen; Feizi, Ten; Soldati-Favre, Dominique

    2010-01-15

    Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for alpha2-3- over alpha2-6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to alpha2-9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6'sulfo-sialyl Lewis(x) might have implications for tissue tropism.

  12. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  13. Effects of environmental factors on C-type lectin recognition to zooxanthellae in the stony coral Pocillopora damicornis.

    Science.gov (United States)

    Zhou, Zhi; Zhao, Shuimiao; Ni, Junyi; Su, Yilu; Wang, Lingui; Xu, Yanlai

    2018-05-15

    C-type lectin is a superfamily of Ca 2+ -dependent carbohydrate-recognition proteins that play significant roles in nonself-recognition and pathogen clearance. In the present study, a C-type lectin (PdC-Lectin) was chosen from stony coral Pocillopora damicornis to understand its recognition characteristics to zooxanthellae. PdC-Lectin protein contained a signal peptide and a carbohydrate-recognition domain with EPN motif in Ca 2+ -binding site 2. The PdC-Lectin recombinant protein was expressed and purified in vitro. The binding of PdC-Lectin protein to zooxanthellae was determined with western blotting method, and the bound protein to 10-10 5  cell mL -1 zooxanthellae was detectable in a concentration-dependent manner. Less PdC-Lectin protein binding to zooxanthellae was observed for the incubation at 36 °C than that at 26 °C. Furthermore, the PAMP recognition spectrum of PdC-Lectin protein was tested through surface plasmon resonance method, and it bound to LPS and Lipid A, but not to LTA, β-glucan, mannose or Poly (I:C). When PdC-Lectin protein was preincubated with LPS, there was less protein binding to zooxanthellae compared with that in non-preincubation group. These results collectively suggest that PdC-Lectin could recognize zooxanthellae, and the recognition could be repressed by high temperature and pathogenic bacteria, which would help to further understand the molecular mechanism of coral bleaching and the establishment of coral-zooxanthella symbiosis in the stony coral P. damicornis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Mannose-binding lectin is a disease modifier in clinical malaria and may function as opsonin for Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

    Garred, Peter; Nielsen, Morten A; Kurtzhals, Jørgen

    2003-01-01

    Variant alleles in the mannose-binding lectin (MBL) gene (mbl2) causing low levels of functional MBL are associated with susceptibility to different infections and are common in areas where malaria is endemic. Therefore, we investigated whether MBL variant alleles in 551 children from Ghana were...... associated with the occurrence and outcome parameters of Plasmodium falciparum malaria and asked whether MBL may function as an opsonin for P. falciparum. No difference in MBL genotype frequency was observed between infected and noninfected children or between children with cerebral malaria and/or severe...... malarial anemia and children with uncomplicated malaria. However, patients with complicated malaria who were homozygous for MBL variant alleles had significantly higher parasite counts and lower blood glucose levels than their MBL-competent counterparts. Distinct calcium-dependent binding of MBL...

  15. Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

    Science.gov (United States)

    Unverzagt, K L; Martinson, J; Lee, W; Stiff, P J; Williams, S; Bender, J G

    1996-01-01

    Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.

  16. Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

    Science.gov (United States)

    Bovi, Michele; Carrizo, Maria E; Capaldi, Stefano; Perduca, Massimiliano; Chiarelli, Laurent R; Galliano, Monica; Monaco, Hugo L

    2011-08-01

    A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.

  17. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  18. Protozoa lectins and their role in host-pathogen interactions.

    Science.gov (United States)

    Singh, Ram Sarup; Walia, Amandeep Kaur; Kanwar, Jagat Rakesh

    2016-01-01

    Lectins are proteins/glycoproteins of non-immune origin that agglutinate red blood cells, lymphocytes, fibroblasts, etc., and bind reversibly to carbohydrates present on the apposing cells. They have at least two carbohydrate binding sites and their binding can be inhibited by one or more carbohydrates. Owing to carbohydrate binding specificity of lectins, they mediate cell-cell interactions and play role in protozoan adhesion and host cell cytotoxicity, thus are central to the pathogenic property of the parasite. Several parasitic protozoa possess lectins which mediate parasite adherence to host cells based on their carbohydrate specificities. These interactions could be exploited for development of novel therapeutics, targeting the adherence and thus helpful in eradicating wide spread of protozoan diseases. The current review highlights the present state knowledge with regard to protozoal lectins with an emphasis on their haemagglutination activity, carbohydrate specificity, characteristics and also their role in pathogenesis notably as adhesion molecules, thereby aiding the pathogen in disease establishment. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. A tick mannose-binding lectin inhibits the vertebrate complement cascade to enhance transmission of the Lyme disease agent

    OpenAIRE

    Schuijt, Tim J.; Coumou, Jeroen; Narasimhan, Sukanya; Dai, Jianfeng; DePonte, Kathleen; Wouters, Diana; Brouwer, Mieke; Oei, Anneke; Roelofs, Joris J.T.H.; van Dam, Alje P.; van der Poll, Tom; van ’t Veer, Cornelis; Hovius, Joppe W.; Fikrig, Erol

    2011-01-01

    The Lyme disease agent, Borrelia burgdorferi, is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8 that reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade resulting in impaired neutrophil phagocytosis and chemotaxis, and diminished Borrelia lysi...

  20. PO-20 - Crosstalk between the lectin pathway and haemostasis in patients with pulmonary cancer

    DEFF Research Database (Denmark)

    Larsen, J B; Christensen, T D; Hvas, C L

    2016-01-01

    INTRODUCTION: Recent research has focused on the complement system in cancer, including the lectin pathway of complement activation. Mannose-binding lectin (MBL), a key activator of the lectin pathway, can bind to tumor cell surfaces in vitro, and lectin pathway activation is increased in several...

  1. Differential effect of plant lectins on mast cells of different origins

    Directory of Open Access Journals (Sweden)

    F.C. Lopes

    2005-06-01

    Full Text Available Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A, lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA, and demetallized Con A and C. brasiliensis, using 1-300 µg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A and on Rowett nude rat (animal free of immunoglobulins peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA. No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars. Additionally, the lectins

  2. Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord

    Science.gov (United States)

    1984-11-30

    MEDICAL CENTER WILFORD HALL AIR FORCE MEDICAL CENTER Title of Thesis: "Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord" Name of...that the use of any copyrighted material in the dissertation manuscript entitled: "Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord...University of the Health Sciences 11 Abstract Title of Thesis: Heterogenity of Opioid Binding Sites In Guinea Pig Spinal Cord Gary Dean Zarr MAJ/ANC

  3. Defining the bacteroides ribosomal binding site.

    Science.gov (United States)

    Wegmann, Udo; Horn, Nikki; Carding, Simon R

    2013-03-01

    The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

  4. T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): Possible involvement in differential recognition of bacteria

    Digital Repository Service at National Institute of Oceanography (India)

    Gowda, N.M.; Goswami, U.; Khan, M.I.

    report a study of a lectin (HSL) involved in immune response in the echinoderm, sea cucumber (Holothuria scabra). Correlative studies indicate that the expression of this defensive lectin is induced by bacterial challenge, wherein cell wall...

  5. Ulex europaeus I and glycine max bind to the human olfactory bulb.

    Science.gov (United States)

    Nagao, M; Oka, N; Kamo, H; Akiguchi, I; Kimura, J

    1993-12-24

    The distribution of binding sites for the fucose-selective lectin Ulex europaeus I and the terminal N-acetylgalactosamine-selective lectin glycine max in the human olfactory bulb were studied. These lectins bound to primary olfactory axons in the olfactory nerve layer and the glomerular layer. They also bound to fibers located in the deeper layers such as the external plexiform layer and the granular layer. Furthermore they projected to the olfactory stalk but not in the cerebrum. The deeper projections of the lectin binding fibers may affect the function of the olfactory bulb in humans.

  6. Association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients

    DEFF Research Database (Denmark)

    Garred, P; Voss, A; Madsen, H O

    2001-01-01

    This study describes the importance of mannose-binding lectin (MBL) variant alleles for systemic lupus erythematosus (SLE) and accompanying infections in a population-based cohort. MBL alleles were determined in 99 SLE patients recruited from a representative Danish region. Patients were classified...... according to the 1982 revised ACR criteria as definite SLE (D-SLE) (n = 77) fulfilling > or =4 criteria and incomplete SLE (I-SLE) (n = 22) with 0.99, respectively). A meta-analysis of eight previously published studies suggested that the presence of MBL variant alleles confer a 1.6 times overall increased...... risk for D-SLE (P disease activity (SLEDAI-index) in a 2-year follow-up period (P = 0.02) and had an increased risk of acquiring complicating infections in general (P = 0.03) and respiratory infections in particular (P = 0.0006). Only in SLE patients...

  7. Biological variation in circulating levels of mannan-binding lectin (MBL) and MBL-associated serine protease-2 and the influence of age, gender and physical exercise

    DEFF Research Database (Denmark)

    Ytting, H; Christensen, IJ; Thiel, Steffen

    2007-01-01

    Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) are central components of the MBL pathway of complement activation, and may have potential as clinical biomarkers in colorectal cancer (CRC). Prior to clinical usage, knowledge of the biological variations of the molecules...... is needed. We here investigate variations of MBL and MASP-2 in healthy persons over time and in relation to gender, age and physical activity. MBL and MASP-2 concentrations were determined in serum from healthy adults over a 3-week period and this was repeated 6 months later (n = 32); during a 24-h period...... not affect the levels (P > 0.8). Serum and plasma levels were only marginally different, and were independent of age and gender. Circulating levels of MBL and MASP-2 are stable over time in healthy individuals, which is advantageous for their potential application as biomarkers....

  8. Biological variation in circulating levels of mannan-binding lectin (MBL) and MBL-associated serine protease-2 and the influence of age, gender and physical exercise

    DEFF Research Database (Denmark)

    Ytting, H; Christensen, I J; Thiel, S

    2007-01-01

    Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) are central components of the MBL pathway of complement activation, and may have potential as clinical biomarkers in colorectal cancer (CRC). Prior to clinical usage, knowledge of the biological variations of the molecules...... is needed. We here investigate variations of MBL and MASP-2 in healthy persons over time and in relation to gender, age and physical activity. MBL and MASP-2 concentrations were determined in serum from healthy adults over a 3-week period and this was repeated 6 months later (n = 32); during a 24-h period...... not affect the levels (P > 0.8). Serum and plasma levels were only marginally different, and were independent of age and gender. Circulating levels of MBL and MASP-2 are stable over time in healthy individuals, which is advantageous for their potential application as biomarkers....

  9. Mannose-binding lectin gene polymorphisms are associated with disease activity and physical disability in untreated, anti-cyclic citrullinated peptide-positive patients with early rheumatoid arthritis

    DEFF Research Database (Denmark)

    Jacobsen, Søren; Garred, Peter; Madsen, Hans Ole

    2009-01-01

    OBJECTIVE: To study the association between polymorphisms in the mannose-binding lectin gene (MBL2) and disease activity, physical disability, and joint erosions in patients with newly diagnosed rheumatoid arthritis (RA). METHODS: Patients with early RA (n=158) not previously treated with disease...... modifying antirheumatic drugs, participating in a treatment trial (CIMESTRA study) were examined at inclusion for MBL2 pooled structural genotypes (O/O, A/O, A/A), regulatory MBL2 promoter polymorphism in position -221 (XX, XY, YY), anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), disease...... activity by Disease Activity Score-28 (DAS28 score), physical disability by Health Assessment Questionnaire (HAQ) score, and erosive changes in hands and feet (Sharp-van der Heijde score). RESULTS: Eight patients were homozygous MBL2 defective (O/O), 101 belonged to an intermediate group, and 49 were MBL2...

  10. Biological Variation in Circulating Levels of Mannan-Binding Lectin (MBL) and MBL-Associated Serine Protease-2 and the Influence of Age, Gender and Physical Exercise

    DEFF Research Database (Denmark)

    Ytting, Henriette; Christensen, Ib Jarle; Thiel, S.

    2007-01-01

    is needed. We here investigate variations of MBL and MASP-2 in healthy persons over time and in relation to gender, age and physical activity. MBL and MASP-2 concentrations were determined in serum from healthy adults over a 3-week period and this was repeated 6 months later (n = 32); during a 24-h period...... not affect the levels (P > 0.8). Serum and plasma levels were only marginally different, and were independent of age and gender. Circulating levels of MBL and MASP-2 are stable over time in healthy individuals, which is advantageous for their potential application as biomarkers.......Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) are central components of the MBL pathway of complement activation, and may have potential as clinical biomarkers in colorectal cancer (CRC). Prior to clinical usage, knowledge of the biological variations of the molecules...

  11. Variant mannose-binding lectin alleles are not associated with susceptibility to or outcome of invasive pneumococcal infection in randomly included patients

    DEFF Research Database (Denmark)

    Kronborg, Gitte; Weis, Nina; Madsen, Hans O

    2002-01-01

    for pneumococcal infections. To assess the influence of MBL genotypes on the course and outcome of invasive pneumococcal disease, clinical data for 141 adult patients were collected prospectively and their genotypes were determined. All patients included had positive blood cultures for Streptococcus pneumoniae....... The distribution of variant MBL alleles related to low MBL serum concentrations was similar among the patients and healthy individuals, and MBL genotype was not associated with infection outcome. Thus, in a random adult population with invasive pneumococcal infection, MBL does not seem to play a role......Invasive pneumococcal disease is a serious infection that primarily affects very young children and elderly or immunocompromised individuals but also affects previously healthy people. Variant mannose-binding lectin (MBL) alleles are associated with recurrent infections and may be a risk factor...

  12. Studies on the relationship between lectin binding carbohydrates and different strains of Leishmania from the New World

    Directory of Open Access Journals (Sweden)

    J. Schottelius

    1982-03-01

    Full Text Available The culture forms of L. mexicana pifanoi (LRC L-90, L. mexicana mexicana (LRC L-94, M-379; L. braziliensis braziliensis (LRC L-77, L-1, M-2903, H-LSS and L. mexicana amazonensis (H-JMMO, M-JOF, H-21, H-PLL,M-1696 were tested with the following lectins: Canavalia ensiformis, Ricinus communis-120, Axinella polypoides, Phaseolus vulgaris, Evonymus europaeus, lotus tetragonolobus, Dolichos biflorus, Aaptos papillata II, Laburnum alpinum, Ulex europaeus, Arachis hypogaea and Soja hispida. All examined strains of Leishmania were agglutinated by C. ensiformis, R. communis-120 and A. popypoides. No agglutination reactions were observed with P. vulgaris, D.biflorus, A. papillata II, E. europaeus and L. tetragonolobus. Only L. m. pifanoi and the L. m. amazonensis strains H-JMMO and MJOF showed agglutination reactions with S. hispida, U. europaeus, L. alpinum and A. hypogaea, while L. m. mexicana (LRC L-94; M-379 strains, L. b. braziliensis H. LSS, LRC L-77; L-1; M-2903 and the L. m. amazonensis strains, H-PLL, H-21, M-1696 showed no agglutination reactions with these four lectins.

  13. Interactions of soybean lectin, soyasaponins, and glycinin with rabbit jejunal mucosa in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, J.R.; Torres-Pinedo, R.

    1982-09-01

    Mucosal samples from rabbit jejunum were incubated (30 min, 25 degrees C) with (/sup 125/I)glycinin in the presence of buffer, soybean lectin (50 micrograms/ml) soyasaponins (1 mg/ml), or both lectin and saponins. The mucosal uptake of (/sup 125/I)glycinin was negligible with buffer, and increased progressively with additions of soybean lectin (P less than 0.05), soyasaponins (P less than 0.005), and both (P less than 0.0001). The stimulation of uptake by lectin and saponins together was greater than the sum of their individual effects (P less than 0.0005). The effect of soybean lectin on glycinin uptake was concentration dependent, reaching a maximum at approximately 50 micrograms/ml for the stimulation of uptake in the presence of saponins, and was inhibited by D-GaINAc. Although the mechanisms involved in mucosal uptake of glycinin cannot be described from these data, we have assumed the presence of two independent pathways for lectin-stimulated and saponin-induced uptakes. In addition, we have proposed that soybean lectin, by binding to terminal galactoside sites at the enterocyte apical membrane, enhances a crenator effect of saponins that leads to increasing leakage of glycinin into the cell.

  14. BEL β-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms.

    Science.gov (United States)

    Bovi, Michele; Cenci, Lucia; Perduca, Massimiliano; Capaldi, Stefano; Carrizo, Maria E; Civiero, Laura; Chiarelli, Laurent R; Galliano, Monica; Monaco, Hugo L

    2013-05-01

    A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

  15. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    Science.gov (United States)

    Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  16. Lectin binders. A new group of plant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rudiger, H; Gebauer, G; Gansera, R; Schurz, H; Schimpl, A [Wuerzburg Univ. (Germany, F.R.)

    1982-09-01

    Lectins are widely distributed in the plant kingdom, many of them being well characterized in their chemical structure and the effects they have on alien biological systems such as erythrocytes or lymphocytes. The biological function of plant lectins remains speculative. We therefore inspected plant extracts from components which might bind specifically to the lectin from the respective plant. Single proteins (lectin binders) could be isolated from each plant extract. The interaction of these proteins with lectins was demonstrated and qualified by several methods. Similar to the lectins, the lectin binders are localized in the cytoplasm in contrast to them, however, they persist during germination and plant growth. Their precise role in the plant is not known, but they are likely to be associated with lectins not only in vitro but also in vivo. They also interact with alien cells, and are able to stimulate mitosis in murine lymphocytes. Some lectin binders act specifically on B lymphocytes, leaving T cells uninfluenced.

  17. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  18. Purification, subunit characterization and ultrastructure of three soluble bovine lectins: conglutinin, mannose-binding protein and the pentraxin serum amyloid P-component

    DEFF Research Database (Denmark)

    Andersen, Ove; Friis, P; Holm Nielsen, E

    1992-01-01

    affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated....... The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding...... of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 k...

  19. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    Science.gov (United States)

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-02-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

  20. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  1. Lectins in human pathogenic fungi.

    Science.gov (United States)

    Gallegos, Belém; Martínez, Ruth; Pérez, Laura; Del Socorro Pina, María; Perez, Eduardo; Hernández, Pedro

    2014-01-01

    Lectins are carbohydrate-binding proteins widely distributed in nature. They constitute a highly diverse group of proteins consisting of many different protein families that are, in general, structurally unrelated. In the last few years, mushroom and other fungal lectins have attracted wide attention due to their antitumour, antiproliferative and immunomodulatory activities. The present mini-review provides concise information about recent developments in understanding lectins from human pathogenic fungi. A bibliographic search was performed in the Science Direct and PubMed databases, using the following keywords "lectin", "fungi", "human" and "pathogenic". Lectins present in fungi have been classified; however, the role played by lectins derived from human pathogenic fungi in infectious processes remains uncertain; thus, this is a scientific field requiring more research. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  2. Differential activity of a lectin from Solieria filiformis against human pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    M.L. Holanda

    2005-12-01

    Full Text Available A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium. The lectin (500 µg/mL stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 µg/mL but the lectin (10-1000 µg/mL had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 µg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 µg/mL, its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.

  3. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  4. Variant G57E of mannose binding lectin associated with protection against tuberculosis caused by Mycobacterium africanum but not by M. tuberculosis.

    Directory of Open Access Journals (Sweden)

    Thorsten Thye

    Full Text Available Structural variants of the Mannose Binding Lectin (MBL cause quantitative and qualitative functional deficiencies, which are associated with various patterns of susceptibility to infectious diseases and other disorders. We determined genetic MBL variants in 2010 Ghanaian patients with pulmonary tuberculosis (TB and 2346 controls and characterized the mycobacterial isolates of the patients. Assuming a recessive mode of inheritance, we found a protective association between TB and the MBL2 G57E variant (odds ratio 0.60, confidence interval 0.4-0.9, P 0.008 and the corresponding LYQC haplotype (P(corrected 0.007 which applied, however, only to TB caused by M. africanum but not to TB caused by M. tuberculosis. In vitro, M. africanum isolates bound recombinant human MBL more efficiently than did isolates of M. tuberculosis. We conclude that MBL binding may facilitate the uptake of M. africanum by macrophages, thereby promoting infection and that selection by TB may have favoured the spread of functional MBL deficiencies in regions endemic for M. africanum.

  5. Exploring Alternative Radiolabeling Strategies for Sialic Acid-Binding Immunoglobulin-Like Lectin 9 Peptide: [68Ga]Ga- and [18F]AlF-NOTA-Siglec-9

    Directory of Open Access Journals (Sweden)

    Olli Moisio

    2018-01-01

    Full Text Available Amino acid residues 283–297 from sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9 form a cyclic peptide ligand targeting vascular adhesion protein-1 (VAP-1. VAP-1 is associated with the transfer of leukocytes from blood to tissues upon inflammation. Therefore, analogs of Siglec-9 peptide are good candidates for visualizing inflammation non-invasively using positron emission tomography (PET. Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA-conjugated Siglec-9 has been evaluated extensively for this purpose. Here, we explored two alternative strategies for radiolabeling Siglec-9 peptide using a 1,4,7-triazacyclononane-triacetic acid (NOTA-chelator to bind [68Ga]Ga or [18F]AlF. The radioligands were evaluated by in vivo PET imaging and ex vivo γ-counting of turpentine-induced sterile skin/muscle inflammation in Sprague-Dawley rats. Both tracers showed clear accumulation in the inflamed tissues. The whole-body biodistribution patterns of the tracers were similar.

  6. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    International Nuclear Information System (INIS)

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-01-01

    Autoradiography using 125 I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat

  7. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-04-01

    Autoradiography using /sup 125/I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat.

  8. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  9. Specific binding of Ulex europaeus agglutinin I lectin to sarcolemma of distal myopathy with rimmed vacuole formation.

    Science.gov (United States)

    Yatabe, K; Kawai, M

    1997-08-01

    Ulex europaeus agglutinin I (UEA I) binding was studied in 83 patients with various neuromuscular disorders. UEA I labelled endomysial capillaries and endothelial cells of perimysial blood vessels in all the examined muscles. There was no UEA I binding to muscle fibres except for all (9) cases of distal myopathy with rimmed vacuole formation (DMRV), 1 of 5 cases of inclusion body myositis and 1 of 36 cases of inflammatory myopathies. The UEA I binding was completely eliminated by preincubation of UEA I solution with L-fucose. Using electron microscopy, the UEA I binding was localized to sarcolemma and intrasarco-plasmic membranous organelles other than mitochondria. Myosatellite cells were not labelled. These findings revealed the existence of fucosylated proteins or lipids in a subset of skeletal muscles suffering from DMRV. Biochemical identification of the fucosylated substance and further detailed study on subcellular localization of UEA I binding may yield important clues to the unknown pathogenesis of DMRV.

  10. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  11. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  12. (-)PPAP: a new and selective ligand for sigma binding sites.

    Science.gov (United States)

    Glennon, R A; Battaglia, G; Smith, J D

    1990-11-01

    Most agents employed for the investigation of sigma (sigma) binding sites display relatively low affinity for these sites, bind both at sigma sites and at either phencyclidine (PCP) sites or dopamine receptors with similar affinity, and/or produce some dopaminergic activity in vivo. We describe a new agent, (-)PPAP or R(-)-N-(3-phenyl-n-propyl)-1-phenyl-2-aminopropane hydrochloride, that binds with high affinity and selectivity at sigma (IC50 = 24 nM) versus either PCP sites (IC50 greater than 75,000 nM) or D1 and D2 dopamine receptors (IC50 greater than 5,000 nM). The sigma affinity of this agent is comparable to that of the standard ligands (+)-3-PPP and DTG. Furthermore, although (-)PPAP is structurally related to amphetamine, it neither produces nor antagonizes amphetamine-like stimulus effect in rats trained to discriminate 1 mg/kg of S(+)amphetamine from saline.

  13. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs...

  14. Structure prediction and functional analysis of a non-permutated lectin from Dioclea grandiflora.

    Science.gov (United States)

    de Sousa, Bruno Lopes; Nagano, Celso Shiniti; Simões, Rafael da Conceição; Silva-Filho, José Caetano; Cunha, Rodrigo Maranguape da Silva; Cajazeiras, João Batista; do Nascimento, Kyria Santiago; Cavada, Benildo Sousa

    2016-12-01

    Legume lectins have been widely studied and applied for many purposes in the last few decades, but many of their physiological aspects remain elusive. The Diocleinae legume subtribe, which includes intensively explored lectins, such as ConA, presents an unusual and extensive post-translational process which results in minor alterations in protein structure, in turn making its function elusive. Despite previous reports about Diocleinae precursor activity, no structural or functional analyses have ever been carried out to understand the impacts of post-translational processing relative to lectin structure and binding specificity. Here we analyzed the functionality of a non glycosylated, recombinantly expressed lectin precursor from Dioclea grandiflora through inhibition assays, corroborating the experimental data with structural information generated by molecular modeling, docking calculations and molecular dynamics simulations. We demonstrate that Diocleinae precursors are active and share the same carbohydrate specificity as mature lectins. At the same time, however, subtle structural alterations were detected and mostly result in an "incomplete" functionality of the precursor, as consequence of an immature binding site and an unstructured tetramer interface, affecting carbohydrate binding and oligomer formation, respectively. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    Science.gov (United States)

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  16. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

    International Nuclear Information System (INIS)

    de Miranda Santos, I.K.; Pereira, M.E.

    1984-01-01

    Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various 125 I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeus and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments

  18. Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

    Energy Technology Data Exchange (ETDEWEB)

    de Miranda Santos, I.K.; Pereira, M.E.

    1984-09-01

    Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeus and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.

  19. Opioid binding site in EL-4 thymoma cell line

    International Nuclear Information System (INIS)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of [ 3 H] bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10 6 cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of [ 3 H] bremazocine with an IC 50 value = 0.57μM. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, [D-Pen 2 , D-Pen 5 ] enkephalin and β-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC 50 = 60μM, that was similar to naloxone. 32 references, 3 figures, 2 tables

  20. Human chorionic ganodotropin binding sites in the human endometrium

    International Nuclear Information System (INIS)

    Bhattacharya, S.; Banerjee, J.; Sen, S.; Manna, P.R.

    1993-01-01

    The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. The authors have identified the hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5x10 -10 mol/l and in anovulatory women to be 3.1x10 -10 mol/l. The maximum binding capacity varied considerably between ovulatory and anovulatory endometrium. Among the divalent metal ions tested Zn 2+ effected a remarkable increase in [ 125 I]hCG binding to the endometrium, whereas Mn 2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [ 125 I]hCG binding to endometrium. 14 refs., 3 figs

  1. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  2. Transcription factor binding sites prediction based on modified nucleosomes.

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    Mohammad Talebzadeh

    Full Text Available In computational methods, position weight matrices (PWMs are commonly applied for transcription factor binding site (TFBS prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, "modified nucleosomes neighboring" and "modified nucleosomes occupancy", to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method

  3. Investigation of Monnose-Binding Lectin gene Polymorphism in Patients with Erythema Multiforme, Stevens-Johnson Syndrome and Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Overlap Syndrome

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    Sevil Toka

    2012-09-01

    Full Text Available Objective: Monnose-Binding lectin (MBL appears to play an important role in the immune system. The genetic polymorphisms in the MBL2 gene can result in a reduction of serum levels, leading to a predisposition to recurrent infection. The aim of this study is to investigate the influence of a polymorphism in codon 54 of the MBL2 gene on the susceptibility to Erythema Multiforme, Stevens-Johnson Syndrome and Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Overlap Syndrome (EM, SJS and SJS/TEN overlap syndrome. Material and Methods: Our study included 64 patients who were clinically and/or histopathologically diagnosed with EM, SJS, and SJS/TEN overlap syndrome and 66 healthy control subjects who were genotyped for the MBL2 gene codon 54 polymorphism using the PCR-RFLP method. For all statistical analyses, the level of significance was set at p<0.05. Results: The prevalence of the B allele was 18% in the EM, SJS and SJS/TEN patient groups and 13% in the control group. No significant differences in allele frequencies of any polymorphism were observed between the patient and control groups, although the B allele was more frequent in the patient groups (p=0.328.Conclusion: Our results provide no evidence of a relationship between MBL2 gene codon 54 polymorphism and the susceptibility to EM, SJS and SJS/TEN overlap syndrome. However, these findings should be confirmed in studies with a larger sample size.

  4. Mannose-binding lectin codon 54 gene polymorphism in relation to risk of nosocomial invasive fungal infection in preterm neonates in the neonatal intensive care unit.

    Science.gov (United States)

    Aydemir, Cumhur; Onay, Huseyin; Oguz, Serife Suna; Ozdemir, Taha Resid; Erdeve, Omer; Ozkinay, Ferda; Dilmen, Ugur

    2011-09-01

    Preterm neonates are susceptible to infection due to a combination of sub-optimal immunity and increased exposure to invasive organisms. Invasive fungal infections are associated with significant morbidity and mortality among preterm infants cared for in the neonatal intensive care unit (NICU). Mannose-binding lectin (MBL) is a component of the innate immune system, which may be especially important in the neonatal setting. The objective of this study was to investigate the presence of any association between MBL gene polymorphism and nosocomial invasive fungal infection in preterm neonates. Codon 54 (B allele) polymorphism in exon 1 of the MBL gene was investigated in 31 patients diagnosed as nosocomial invasive fungal infection and 30 control preterm neonates. AB genotype was determined in 26% and 30% of patient and control groups, respectively, and the difference was not statistically significant. AA genotype was determined in 74% of the patient group and in 67% of the control group, and the difference was not statistically significant. B allele frequency was not different significantly in the patient group (13%) compared to the control group (18%). In our study, no relationship was found between MBL codon 54 gene polymorphism and the risk of nosocomial invasive fungal infection in preterm neonates in NICU.

  5. Association study of genetic variants at single nucleotide polymorphism rs109231409 of mannose-binding lectins 1 gene with mastitis susceptibility in Vrindavani crossbred cattle

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    V. N. Muhasin Asaf

    2014-10-01

    Full Text Available Aim: The present study was undertaken to identify whether single nucleotide polymorphism (SNP rs109231409 located on mannose-binding lectins 1 (MBL1 gene was associated with mastitis tolerance/susceptibility. Materials and Methods: After grouping 100 Vrindavani crossbred cattle as mastitis positive and negative animals, they were genotyped using polymerase chain reaction (PCR-restriction fragment length polymorphisms method. Gene and genotype frequencies of different patterns were estimated by standard procedure (POPGENE version 1.32, (University of Alberta, Canada and statistical analysis was carried out by logistic regression methods using STATA 12 software (StataCorp LP, USA. Results: The 588 bp fragment of MBL1 gene was amplified using PCR. PCR product was digested with ApaI restriction enzyme showed two distinct genotypes viz., GG (311 bp and 272 bp fragments and GA (588 bp, 311 bp and 277 bp fragments. The gene, genotype frequencies, average heterozygosity, polymorphic information content and χ2 values for the locus rs109231409 was ascertained. Conclusions: No significant association between SNP “rs109231409” with mastitis tolerance was found. Although there is a lack of association, further studies have to be undertaken in a large population in order to validate the impact of rs109231409 (g.855G >A on mastitis tolerance.

  6. Mannose-binding lectin 2 (Mbl2 gene polymorphisms are related to protein plasma levels, but not to heart disease and infection by Chlamydia

    Directory of Open Access Journals (Sweden)

    M.A.F. Queiroz

    Full Text Available The presence of the single nucleotide polymorphisms in exon 1 of the mannose-binding lectin 2 (MBL2 gene was evaluated in a sample of 159 patients undergoing coronary artery bypass surgery (71 patients undergoing valve replacement surgery and 300 control subjects to investigate a possible association between polymorphisms and heart disease with Chlamydia infection. The identification of the alleles B and D was performed using real time polymerase chain reaction (PCR and of the allele C was accomplished through PCR assays followed by digestion with the restriction enzyme. The comparative analysis of allelic and genotypic frequencies between the three groups did not reveal any significant difference, even when related to previous Chlamydia infection. Variations in the MBL plasma levels were influenced by the presence of polymorphisms, being significantly higher in the group of cardiac patients, but without representing a risk for the disease. The results showed that despite MBL2 gene polymorphisms being associated with the protein plasma levels, the polymorphisms were not enough to predict the development of heart disease, regardless of infection with both species of Chlamydia.

  7. Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

    2016-07-08

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

  8. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  9. Lectin histochemistry of goblet cell sugar residues in the gut of the chick embryo and of the newborn.

    Science.gov (United States)

    Bryk, S G; Sgambati, E; Gheri Bryk, G

    1999-04-01

    The anlage of duodenum, ileum and colon were removed from chick embryos of day 8-21 of incubation and from 1-day-old chicks. A battery of seven different horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, Con A, WGA, LTA and UEAI) was used to study the carbohydrate residues of the glycoconjugates in the goblet cells of the three parts of the intestine. The main results can be summarized as follows: differences in lectin binding were absent in the proximal and distal parts of the duodenum, ileum and colon. Lectin histochemistry showed differences among the three intestinal segments for the time of appearance of the oligosaccharides in the goblet mucus. In the colonic goblet cells of 1-day-old chicks, LTA and UEAI lectins showed two different types of linkage of alpha-L-fucose. This is the first demonstration of UEAI reactive sites in Gallus domesticus.

  10. Probing binding hot spots at protein-RNA recognition sites.

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-29

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Regulation of ozone-induced lung inflammation and injury by the β-galactoside-binding lectin galectin-3

    International Nuclear Information System (INIS)

    Sunil, Vasanthi R.; Francis, Mary; Vayas, Kinal N.; Cervelli, Jessica A.; Choi, Hyejeong; Laskin, Jeffrey D.; Laskin, Debra L.

    2015-01-01

    Macrophages play a dual role in ozone toxicity, contributing to both pro- and anti-inflammatory processes. Galectin-3 (Gal-3) is a lectin known to regulate macrophage activity. Herein, we analyzed the role of Gal-3 in the response of lung macrophages to ozone. Bronchoalveolar lavage (BAL) and lung tissue were collected 24–72 h after exposure (3 h) of WT and Gal-3 -/- mice to air or 0.8 ppm ozone. In WT mice, ozone inhalation resulted in increased numbers of proinflammatory (Gal-3 + , iNOS + ) and anti-inflammatory (MR-1 + ) macrophages in the lungs. While accumulation of iNOS + macrophages was attenuated in Gal-3 -/- mice, increased numbers of enlarged MR-1 + macrophages were noted. This correlated with increased numbers of macrophages in BAL. Flow cytometric analysis showed that these cells were CD11b + and consisted mainly (> 97%) of mature (F4/80 + CD11c + ) proinflammatory (Ly6GLy6C hi ) and anti-inflammatory (Ly6GLy6C lo ) macrophages. Increases in both macrophage subpopulations were observed following ozone inhalation. Loss of Gal-3 resulted in a decrease in Ly6C hi macrophages, with no effect on Ly6C lo macrophages. CD11b + Ly6G + Ly6C + granulocytic (G) and monocytic (M) myeloid derived suppressor cells (MDSC) were also identified in the lung after ozone. In Gal-3 -/- mice, the response of G-MDSC to ozone was attenuated, while the response of M-MDSC was heightened. Changes in inflammatory cell populations in the lung of ozone treated Gal-3 -/- mice were correlated with reduced tissue injury as measured by cytochrome b5 expression. These data demonstrate that Gal-3 plays a role in promoting proinflammatory macrophage accumulation and toxicity in the lung following ozone exposure. - Highlights: • Multiple monocytic-macrophage subpopulations accumulate in the lung after ozone inhalation. • Galectin-3 plays a proinflammatory role in ozone-induced lung injury. • In the absence of gal-3, inflammatory cells with a myeloid derived suppressor cell phenotype

  12. Regulation of ozone-induced lung inflammation and injury by the β-galactoside-binding lectin galectin-3

    Energy Technology Data Exchange (ETDEWEB)

    Sunil, Vasanthi R., E-mail: sunilva@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ (United States); Francis, Mary, E-mail: maryfranrutgers@gmail.com [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ (United States); Vayas, Kinal N., E-mail: kinalv5@gmail.com [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ (United States); Cervelli, Jessica A., E-mail: j.cervelli@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ (United States); Choi, Hyejeong, E-mail: choi@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ (United States)

    2015-04-15

    Macrophages play a dual role in ozone toxicity, contributing to both pro- and anti-inflammatory processes. Galectin-3 (Gal-3) is a lectin known to regulate macrophage activity. Herein, we analyzed the role of Gal-3 in the response of lung macrophages to ozone. Bronchoalveolar lavage (BAL) and lung tissue were collected 24–72 h after exposure (3 h) of WT and Gal-3{sup -/-} mice to air or 0.8 ppm ozone. In WT mice, ozone inhalation resulted in increased numbers of proinflammatory (Gal-3{sup +}, iNOS{sup +}) and anti-inflammatory (MR-1{sup +}) macrophages in the lungs. While accumulation of iNOS{sup +} macrophages was attenuated in Gal-3{sup -/-} mice, increased numbers of enlarged MR-1{sup +} macrophages were noted. This correlated with increased numbers of macrophages in BAL. Flow cytometric analysis showed that these cells were CD11b{sup +} and consisted mainly (> 97%) of mature (F4/80{sup +}CD11c{sup +}) proinflammatory (Ly6GLy6C{sup hi}) and anti-inflammatory (Ly6GLy6C{sup lo}) macrophages. Increases in both macrophage subpopulations were observed following ozone inhalation. Loss of Gal-3 resulted in a decrease in Ly6C{sup hi} macrophages, with no effect on Ly6C{sup lo} macrophages. CD11b{sup +}Ly6G{sup +}Ly6C{sup +} granulocytic (G) and monocytic (M) myeloid derived suppressor cells (MDSC) were also identified in the lung after ozone. In Gal-3{sup -/-} mice, the response of G-MDSC to ozone was attenuated, while the response of M-MDSC was heightened. Changes in inflammatory cell populations in the lung of ozone treated Gal-3{sup -/-} mice were correlated with reduced tissue injury as measured by cytochrome b5 expression. These data demonstrate that Gal-3 plays a role in promoting proinflammatory macrophage accumulation and toxicity in the lung following ozone exposure. - Highlights: • Multiple monocytic-macrophage subpopulations accumulate in the lung after ozone inhalation. • Galectin-3 plays a proinflammatory role in ozone-induced lung injury. • In the

  13. Binding of the mannose-specific lectin, Griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available development. Anti- microb. Agents Chemother. 41:1521?1530. 7. Buchacher, A., et al. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization...

  14. Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL in the mouse model of vaccination against neosporosis

    Directory of Open Access Journals (Sweden)

    Cardoso Mariana R D

    2012-10-01

    Full Text Available Abstract Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA, ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80% and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.

  15. Penicillin-binding site on the Escherichia coli cell envelope

    International Nuclear Information System (INIS)

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-01-01

    The binding of 35 S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

  16. Mannose-binding lectin contributes to deleterious inflammatory response in pandemic H1N1 and avian H9N2 infection.

    Science.gov (United States)

    Ling, Man To; Tu, Wenwei; Han, Yan; Mao, Huawei; Chong, Wai Po; Guan, Jing; Liu, Ming; Lam, Kwok Tai; Law, Helen K W; Peiris, J S Malik; Takahashi, K; Lau, Yu Lung

    2012-01-01

    Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response.

  17. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

    2017-01-01

    is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super......-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate...

  18. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.G.

    1987-01-01

    The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics

  19. Radiotracers for per studies of neurotransmitter binding sites: Design considerations

    International Nuclear Information System (INIS)

    Kilbourn, M.R.

    1991-01-01

    Neurotransmitter binding sites, such as receptors, neuronal uptake systems, and vesicular uptake systems, are important targets for new radiopharmaceutical design. Selection of potential radioligands can be guided by in vitro laboratory data including such characteristics as selectivity and affinity for specific binding sites. However, development of PET radiotracers for use in vivo must include considerations of in vivo pharmacokinetics and metabolism. Introduction of potential radioligands is further narrowed by the demands of the radiochemical synthesis, which must produce radioligands of high chemical and radiochemical purity and of high specific activity. This paper will review examples of previous and current attempts by radiopharmaceutical chemists to meet these demands for new positron emitter-labeled radioligands for PET studies of a wide array of neurotransmitter binding sites

  20. Engineering of PA-IIL lectin from Pseudomonas aeruginosa – Unravelling the role of the specificity loop for sugar preference

    Directory of Open Access Journals (Sweden)

    Imberty Anne

    2007-06-01

    Full Text Available Abstract Background Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose – a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. Results In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography and functionally (by isothermal titration calorimetry. The mutated amino acids (22–23–24 triad belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions – both of afore mentioned have strong effects on the saccharide preferences. Conclusion Mutagenesis of amino acids forming the specificity binding loop allowed

  1. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Multiple [3H]-nemonapride binding sites in calf brain.

    Science.gov (United States)

    Helmeste, D M; Tang, S W; Li, M; Fang, H

    1997-07-01

    [3H]-Nemonapride has been the ligand of choice to label D4 dopamine receptors. Its specificity was questioned when it was discovered that sigma (sigma) sites were also labeled by [3H]-nemonapride. To further characterize the binding of [3H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [3H]-nemonapride labeled multiple sites. Dopaminergic and sigma sites were the most prominent. The sigma binding profile was sigma-1 like with a Ki binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and sigma sites, respectively, revealed additional, not previously characterized binding sites for [3H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [3H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met.

  3. Binding-site assessment by virtual fragment screening.

    Directory of Open Access Journals (Sweden)

    Niu Huang

    2010-04-01

    Full Text Available The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

  4. Eel calcitonin binding site distribution and antinociceptive activity in rats

    International Nuclear Information System (INIS)

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-01-01

    The distribution of binding site for [ 125 I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [ 125 I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain

  5. Structural insights into the anti-HIV activity of the Oscillatoria agardhii agglutinin homolog lectin family.

    Science.gov (United States)

    Koharudin, Leonardus M I; Kollipara, Sireesha; Aiken, Christopher; Gronenborn, Angela M

    2012-09-28

    Oscillatoria agardhii agglutinin homolog (OAAH) proteins belong to a recently discovered lectin family. All members contain a sequence repeat of ~66 amino acids, with the number of repeats varying among different family members. Apart from data for the founding member OAA, neither three-dimensional structures, information about carbohydrate binding specificities, nor antiviral activity data have been available up to now for any other members of the OAAH family. To elucidate the structural basis for the antiviral mechanism of OAAHs, we determined the crystal structures of Pseudomonas fluorescens and Myxococcus xanthus lectins. Both proteins exhibit the same fold, resembling the founding family member, OAA, with minor differences in loop conformations. Carbohydrate binding studies by NMR and x-ray structures of glycan-lectin complexes reveal that the number of sugar binding sites corresponds to the number of sequence repeats in each protein. As for OAA, tight and specific binding to α3,α6-mannopentaose was observed. All the OAAH proteins described here exhibit potent anti-HIV activity at comparable levels. Altogether, our results provide structural details of the protein-carbohydrate interaction for this novel lectin family and insights into the molecular basis of their HIV inactivation properties.

  6. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  7. Mushroom Lectins: Specificity, Structure and Bioactivity Relevant to Human Disease

    Directory of Open Access Journals (Sweden)

    Mohamed Ali Abol Hassan

    2015-04-01

    Full Text Available Lectins are non-immunoglobulin proteins that bind diverse sugar structures with a high degree of selectivity. Lectins play crucial role in various biological processes such as cellular signaling, scavenging of glycoproteins from the circulatory system, cell–cell interactions in the immune system, differentiation and protein targeting to cellular compartments, as well as in host defence mechanisms, inflammation, and cancer. Among all the sources of lectins, plants have been most extensively studied. However, more recently fungal lectins have attracted considerable attention due to their antitumor, antiproliferative and immunomodulatory activities. Given that only 10% of mushroom species are known and have been taxonomically classified, mushrooms represent an enormous unexplored source of potentially useful and novel lectins. In this review we provide an up-to-date summary on the biochemical, molecular and structural properties of mushroom lectins, as well as their versatile applications specifically focusing on mushroom lectin bioactivity.

  8. Dangerous connections : on binding site models of infectious disease dynamics

    NARCIS (Netherlands)

    Leung, Ka Yin; Diekmann, Odo

    2017-01-01

    We formulate models for the spread of infection on networks that are amenable to analysis in the large population limit. We distinguish three different levels: (1) binding sites, (2) individuals, and (3) the population. In the tradition of physiologically structured population models, the

  9. Fabrication of supramolecular frameworks by tuning the binding site ...

    Indian Academy of Sciences (India)

    Administrator

    Fabrication of supramolecular frameworks by tuning the binding site of a tripodal ligand with d. 10 metal ions 803. Table 1. Crystal data and structure refinement parameters for 1 and 2. 1 .... e-mail: deposit@ccdc.cam.ac.uk web: http://www. ccdc. cam.ac.uk/deposit]. Supplementary figures and tables can be found in website ...

  10. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  11. Incorporating evolution of transcription factor binding sites into ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Identifying transcription factor binding sites (TFBSs) is essential to elucidate ... alignments with parts annotated as gap lessly aligned TFBSs (pair-profile hits) are generated. Moreover, the pair- profile related parameters are derived in a sound statistical framework. ... Much research has gone into the study of the evolution of.

  12. Effects of Lectins on initial attachment of cariogenic Streptococcus mutans.

    Science.gov (United States)

    Ito, Takashi; Yoshida, Yasuhiro; Shiota, Yasuyoshi; Ito, Yuki; Yamamoto, Tadashi; Takashiba, Shogo

    2018-02-01

    Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.

  13. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

    Science.gov (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

    2014-02-10

    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  15. [Adenylate cyclase from rabbit heart: substrate binding site].

    Science.gov (United States)

    Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

    1981-08-01

    The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

  16. Bifunctional avidin with covalently modifiable ligand binding site.

    Directory of Open Access Journals (Sweden)

    Jenni Leppiniemi

    Full Text Available The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (streptavidin to improve the existing applications. Even so, (streptavidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

  17. The effects of GH and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein D and vitamin D binding protein in Turner syndrome

    DEFF Research Database (Denmark)

    Gravholt, Claus Højbjerg; Leth-Larsen, Rikke; Lauridsen, Anna Lis

    2004-01-01

    function. In the present study we examined whether GH or hormone replacement therapy (HRT) in Turner syndrome (TS) influence the serum concentrations of MBL and two other proteins partaking in the innate immune defence, surfactant protein D (SP-D) and vitamin D binding protein (DBP). DESIGN: Study 1...

  18. Lessons learned from mice deficient in lectin complement pathway molecules

    DEFF Research Database (Denmark)

    Genster, Ninette; Takahashi, Minoru; Sekine, Hideharu

    2014-01-01

    in turn activate downstream complement components, ultimately leading to elimination of the pathogen. Mice deficient in the key molecules of lectin pathway of complement have been generated in order to build knowledge of the molecular mechanisms of the lectin pathway in health and disease. Despite......The lectin pathway of the complement system is initiated when the pattern-recognition molecules, mannose-binding lectin (MBL), ficolins or collectin-11, bind to invading pathogens or damaged host cells. This leads to activation of MBL/ficolin/collectin-11 associated serine proteases (MASPs), which...... differences in the genetic arrangements of murine and human orthologues of lectin pathway molecules, the knockout mice have proven to be valuable models to explore the effect of deficiency states in humans. In addition, new insight and unexpected findings on the diverse roles of lectin pathway molecules...

  19. Phosphorus Binding Sites in Proteins: Structural Preorganization and Coordination

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Greisen, Per Junior; Junker, Märta Caroline

    2014-01-01

    to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced...... by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell...

  20. The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

    International Nuclear Information System (INIS)

    Mor, Visesato; Chatterjee, Tapati

    2007-01-01

    Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids- 1 A D T I V A V E L D T Y P N 14 -correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A

  1. Opioid binding site in EL-4 thymoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  2. Binding Sites for Amyloid-β Oligomers and Synaptic Toxicity

    Science.gov (United States)

    Smith, Levi M.; Strittmatter, Stephen M.

    2017-01-01

    In Alzheimer’s disease (AD), insoluble and fibrillary amyloid-β (Aβ) peptide accumulates in plaques. However, soluble Aβ oligomers are most potent in creating synaptic dysfunction and loss. Therefore, receptors for Aβ oligomers are hypothesized to be the first step in a neuronal cascade leading to dementia. A number of cell-surface proteins have been described as Aβ binding proteins, and one or more are likely to mediate Aβ oligomer toxicity in AD. Cellular prion protein (PrPC) is a high-affinity Aβ oligomer binding site, and a range of data delineates a signaling pathway leading from Aβ complexation with PrPC to neuronal impairment. Further study of Aβ binding proteins will define the molecular basis of this crucial step in AD pathogenesis. PMID:27940601

  3. Recent Progress in Lectin-Based Biosensors

    Directory of Open Access Journals (Sweden)

    Baozhen Wang

    2015-12-01

    Full Text Available This article reviews recent progress in the development of lectin-based biosensors used for the determination of glucose, pathogenic bacteria and toxins, cancer cells, and lectins. Lectin proteins have been widely used for the construction of optical and electrochemical biosensors by exploiting the specific binding affinity to carbohydrates. Among lectin proteins, concanavalin A (Con A is most frequently used for this purpose as glucose- and mannose-selective lectin. Con A is useful for immobilizing enzymes including glucose oxidase (GOx and horseradish peroxidase (HRP on the surface of a solid support to construct glucose and hydrogen peroxide sensors, because these enzymes are covered with intrinsic hydrocarbon chains. Con A-modified electrodes can be used as biosensors sensitive to glucose, cancer cells, and pathogenic bacteria covered with hydrocarbon chains. The target substrates are selectively adsorbed to the surface of Con A-modified electrodes through strong affinity of Con A to hydrocarbon chains. A recent topic in the development of lectin-based biosensors is a successful use of nanomaterials, such as metal nanoparticles and carbon nanotubes, for amplifying output signals of the sensors. In addition, lectin-based biosensors are useful for studying glycan expression on living cells.

  4. The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.

    Science.gov (United States)

    Ortiz-Urda, Susana; Elbe-Bürger, Adelheid; Smolle, Josef; Marquart, Yvonne; Chudnovsky, Yakov; Ridky, Todd W; Bernstein, Pamela; Wolff, Klaus; Rappersberger, Klemens

    2003-12-01

    Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients' sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.

  5. Carbohydrate Detection and Lectin Isolation from Tegumental Tissue of Fasciola hepatica

    Directory of Open Access Journals (Sweden)

    MB Molaei Rad

    2010-02-01

    Full Text Available "nBackground: Fascioliasis is a chronic hepatic disease and may be resulted from mechani­cal/molecular parasite adhesion to host liver tissue. The aim of this study was to detect surface car­bohydrate and lectin, carbohydrate-binding protein isolation that might be responsible of this molecular binding."nMethods: The present experimental work was conducted in the Department of Medical Parasitol­ogy and Mycology, School of Public Health, Tehran University of Medical Sciences, Te­hran, Iran.  Fasciola hepatica parasites were collected from abattoir (Saman, Tehran, Iran and surface mannose-carbohydrate was detected by fluorescein isothiocyanate (FITC conju­gated lectin (Lentil. Lectin of tegumental tissue from F. hepatica was isolated by affinity chroma­tography and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE."nResults: Mannose carbohydrate was observed on the surface of tegumental tissue from para­site under fluorescence microscope. Carbohydrate-binding protein or lectin with MW of 50 kDa also was isolated from homogenized tegument of helminth."nConclusion: These results are important for understanding of molecular pathogenesis of F. hepat­ica at the chronic phase of fascioliasis

  6. Visualization of specific binding sites of benzodiazepine in human brain

    International Nuclear Information System (INIS)

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-01-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

  7. SP-A binding sites on bovine alveolar macrophages.

    Science.gov (United States)

    Plaga, S; Plattner, H; Schlepper-Schaefer, J

    1998-11-25

    Surfactant protein A (SP-A) binding to bovine alveolar macrophages was examined in order to characterize SP-A binding proteins on the cell surface and to isolate putative receptors from these cells that could be obtained in large amounts. Human SP-A, unlabeled or labeled with gold particles, was bound to freshly isolated macrophages and analyzed with ELISA or the transmission electron microscope. Binding of SP-A was inhibited by Ca2+ chelation, by an excess of unlabeled SP-A, or by the presence of 20 mg/ml mannan. We conclude that bovine alveolar macrophages expose binding sites for SP-A that are specific and that depend on Ca2+ and on mannose residues. For isolation of SP-A receptors with homologous SP-A as ligand we isolated SP-A from bovine lung lavage. SDS-PAGE analysis of the purified SP-A showed a protein of 32-36 kDa. Functional integrity of the protein was demonstrated. Bovine SP-A bound to Dynabeads was used to isolate SP-A binding proteins. From the fractionated and blotted proteins of the receptor preparation two proteins bound SP-A in a Ca2+-dependent manner, a 40-kDa protein showing mannose dependency and a 210-kDa protein, showing no mannose sensitivity. Copyright 1998 Academic Press.

  8. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2010-03-01

    Full Text Available The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates.We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data.Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct-interaction detection and TFBS-discovery accuracy. We estimated the accuracy

  9. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  10. Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.

    1985-01-01

    Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [ 125 I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis

  11. Receptor-ligand binding sites and virtual screening.

    Science.gov (United States)

    Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

    2006-01-01

    Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

  12. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  13. Lectin receptors in the human cornea.

    Science.gov (United States)

    Holmes, M J; Mannis, M J; Lund, J; Jacobs, L

    Five different biotin labeled lectins, Concanavalin-A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA1), Ulex europaeus agglutinin (UEA1), and soybean agglutinin (SBA) were used to study lectin receptors on formalin-fixed paraffin embedded human corneas. Con A stained the cytoplasm, cell, and nuclear membranes of the epithelial cells and stained the stroma diffusely. WGA stained the superficial epithelial cells, the epithelial cell membranes, and the keratocytes of the stroma. SBA did not react with any of the corneal layers. RCA1 heavily stained the keratocytes but did not stain the epithelium. UEA1 lightly stained the epithelial cell cytoplasm and interstitial stroma. All staining reactions could be abolished by omission of the lectin or by the use of the appropriate inhibitory sugar. The lectin binding patterns reported here provide a means for further investigation of carbohydrate structures in the human cornea in both normal and disease states.

  14. Fungal lectin MpL enables entry of protein drugs into cancer cells and their subcellular targeting.

    Science.gov (United States)

    Å Urga, Simon; Nanut, Milica Perišić; Kos, Janko; Sabotič, Jerica

    2017-04-18

    Lectins have been recognized as promising carrier molecules for targeted drug delivery. They specifically bind carbohydrate moieties on cell membranes and trigger cell internalization. Fungal lectin MpL (Macrolepiota procera lectin) does not provoke cancer cell cytotoxicity but is able to bind aminopeptidase N (CD13) and integrin α3β1, two glycoproteins that are overexpressed on the membrane of tumor cells. Upon binding, MpL is endocytosed in a clathrin-dependent manner and accumulates initially in the Golgi apparatus and, finally, in the lysosomes. For effective binding and internalization a functional binding site on the α-repeat is needed. To test the potential of MpL as a carrier for delivering protein drugs to cancer cells we constructed fusion proteins consisting of MpL and the cysteine peptidase inhibitors cystatin C and clitocypin. The fused proteins followed the same endocytic route as the unlinked MpL. Peptidase inhibitor-MpL fusions impaired both the intracellular degradation of extracellular matrix and the invasiveness of cancer cells. MpL is thus shown in vitro to be a lectin that can enable protein drugs to enter cancer cells, enhance their internalization and sort them to lysosomes and the Golgi apparatus.

  15. The neck region of the C-type lectin DC-SIGN regulates its surface spatiotemporal organization and virus-binding capacity on antigen presenting cells

    NARCIS (Netherlands)

    Manzo, C.; Torreno-Pina, J.A.; Joosten, B.; Reinieren-Beeren, I.; Gualda, E.J.; Loza-Alvarez, P.; Figdor, Carl; Garcia Parajo, M.F.; Cambi, A.

    2012-01-01

    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and

  16. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells

    NARCIS (Netherlands)

    Manzo, C.; Torreno-Pina, J.A.; Joosten, B.; Reinieren-Beeren, I.; Gualda, E.J.; Loza-Alvarez, P.; Figdor, C.G.; Garcia-Parajo, M.F.; Cambi, A.

    2012-01-01

    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and

  17. Chromatin immunoprecipitation to analyze DNA binding sites of HMGA2.

    Directory of Open Access Journals (Sweden)

    Nina Winter

    Full Text Available BACKGROUND: HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment procedure two consensus sequences for HMGA2 binding have been identified. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. CONCLUSION: After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

  18. Activation of the lectin complement pathway on human renal ...

    African Journals Online (AJOL)

    This study aimed to investigate the roles of high glucose and mannose-binding lectin (MBL) on the activation of the lectin complement pathway (LCP) on human renal glomerular endothelial cells (HRGECs) in vitro. Flow cytometry analysis, immunofluorescence staining and Western blot were used to detect the cell surface ...

  19. Microbial F-type lectin domains with affinity for blood group antigens.

    Science.gov (United States)

    Mahajan, Sonal; Khairnar, Aasawari; Bishnoi, Ritika; Ramya, T N C

    2017-09-23

    F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis b motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    International Nuclear Information System (INIS)

    Lummis, S.C.R.; Johnston, G.A.R.; Nicoletti, G.; Holan, G.

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial [ 3 H]diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli [ 3 H]diazepam binding are those that are active in displacing [ 3 H]benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed

  1. Where's water? The many binding sites of hydantoin.

    Science.gov (United States)

    Gruet, Sébastien; Pérez, Cristóbal; Steber, Amanda L; Schnell, Melanie

    2018-02-21

    Prebiotic hydantoin and its complexes with one and two water molecules are investigated using high-resolution broadband rotational spectroscopy in the 2-8 GHz frequency range. The hyperfine structure due to the nuclear quadrupole coupling of the two 14 N atoms is analysed for the monomer and the complexes. This characteristic hyperfine structure will support a definitive assignment from low frequency radioastronomy data. Experiments with H 2 18 O provide accurate experimental information on the preferred binding sites of water, which are compared with quantum-chemically calculated coordinates. In the 2-water complexes, the water molecules bind to hydantoin as a dimer instead of individually, indicating the strong water-water interactions. This information provides first insight on how hydantoin interacts with water on the molecular level.

  2. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  3. Cation binding at the node of Ranvier: I. Localization of binding sites during development.

    Science.gov (United States)

    Zagoren, J C; Raine, C S; Suzuki, K

    1982-06-17

    Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

  4. The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

    Directory of Open Access Journals (Sweden)

    Richard Beatson

    Full Text Available Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr and STn (NeuAcα2,6GalNAc-Ser/Thr. These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

  5. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  6. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  7. A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the lyme disease agent.

    Science.gov (United States)

    Schuijt, Tim J; Coumou, Jeroen; Narasimhan, Sukanya; Dai, Jianfeng; Deponte, Kathleen; Wouters, Diana; Brouwer, Mieke; Oei, Anneke; Roelofs, Joris J T H; van Dam, Alje P; van der Poll, Tom; Van't Veer, Cornelis; Hovius, Joppe W; Fikrig, Erol

    2011-08-18

    The Lyme disease agent Borrelia burgdorferi is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8, which reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade, resulting in impaired neutrophil phagocytosis and chemotaxis and diminished Borrelia lysis. Therefore, P8 was renamed the tick salivary lectin pathway inhibitor (TSLPI). TSLPI-silenced ticks, or ticks exposed to TSLPI-immune mice, were hampered in Borrelia transmission. Moreover, Borrelia acquisition and persistence in tick midguts was impaired in ticks feeding on TSLPI-immunized, B. burgdorferi-infected mice. Together, our findings suggest an essential role for the lectin complement cascade in Borrelia eradication and demonstrate how a vector-borne pathogen co-opts a vector protein to facilitate early mammalian infection and vector colonization. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  9. Atrial natriuretic factor binding sites in experimental congestive heart failure

    International Nuclear Information System (INIS)

    Bianchi, C.; Thibault, G.; Wrobel-Konrad, E.; De Lean, A.; Genest, J.; Cantin, M.

    1989-01-01

    A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor [(Ser99, Tyr126)ANF] binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF [des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2] (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease

  10. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    International Nuclear Information System (INIS)

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-01-01

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme

  11. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  12. DeepSite: protein-binding site predictor using 3D-convolutional neural networks.

    Science.gov (United States)

    Jiménez, J; Doerr, S; Martínez-Rosell, G; Rose, A S; De Fabritiis, G

    2017-10-01

    An important step in structure-based drug design consists in the prediction of druggable binding sites. Several algorithms for detecting binding cavities, those likely to bind to a small drug compound, have been developed over the years by clever exploitation of geometric, chemical and evolutionary features of the protein. Here we present a novel knowledge-based approach that uses state-of-the-art convolutional neural networks, where the algorithm is learned by examples. In total, 7622 proteins from the scPDB database of binding sites have been evaluated using both a distance and a volumetric overlap approach. Our machine-learning based method demonstrates superior performance to two other competitive algorithmic strategies. DeepSite is freely available at www.playmolecule.org. Users can submit either a PDB ID or PDB file for pocket detection to our NVIDIA GPU-equipped servers through a WebGL graphical interface. gianni.defabritiis@upf.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  13. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    DEFF Research Database (Denmark)

    Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael

    2015-01-01

    the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy......Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present...... and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity...

  14. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  15. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    International Nuclear Information System (INIS)

    Singh, S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 (angstrom) above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational

  16. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    International Nuclear Information System (INIS)

    Gates, T.S.; Zimmerman, R.P.; Mantyh, C.R.; Vigna, S.R.; Maggio, J.E.; Welton, M.L.; Passaro, E.P. Jr.; Mantyh, P.W.

    1988-01-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for 125 I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells

  17. "Click" saccharide/beta-lactam hybrids for lectin inhibition.

    Science.gov (United States)

    Palomo, Claudio; Aizpurua, Jesus M; Balentová, Eva; Azcune, Itxaso; Santos, J Ignacio; Jiménez-Barbero, Jesús; Cañada, Javier; Miranda, José Ignacio

    2008-06-05

    Hybrid glycopeptide beta-lactam mimetics designed to bind lectins or carbohydrate recognition domains in selectins have been prepared according to a "shape-modulating linker" design. This approach was implemented using the azide-alkyne "click" cycloaddition reaction, and as shown by NMR/MD experiments, binding of the resulting mimetics to Ulex Europaeus Lectin-1 (UEL-1) occurred after a "bent-to-extended" conformational change around a partially rotatable triazolylmethylene moiety.

  18. Microglial Lectins in Health and Neurological Diseases

    Directory of Open Access Journals (Sweden)

    Jian Jing Siew

    2018-05-01

    Full Text Available Microglia are the innate sentinels of the central nervous system (CNS and are responsible for the homeostasis and immune defense of the CNS. Under the influence of the local environment and cell-cell interaction, microglia exhibit a multidimensional and context-dependent phenotypes that can be cytotoxic and neuroprotective. Recent studies suggest that microglia express multitudinous types of lectins, including galectins, Siglecs, mannose-binding lectins (MBLs and other glycan binding proteins. Because most studies that examine lectins focus on the peripheral system, the functions of lectins have not been critically investigated in the CNS. In addition, the types of brain cells that contribute to the altered levels of lectins present in diseases are often unclear. In this review, we will discuss how galectins, Siglecs, selectins and MBLs contribute to the dynamic functions of microglia. The interacting ligands of these lectins are complex glycoconjugates, which consist of glycoproteins and glycolipids that are expressed on microglia or surrounding cells. The current understanding of the heterogeneity and functions of glycans in the brain is limited. Galectins are a group of pleotropic proteins that recognize both β-galactoside-containing glycans and non- β-galactoside-containing proteins. The function and regulation of galectins have been implicated in immunomodulation, neuroinflammation, apoptosis, phagocytosis and oxidative bursts. Most Siglecs are expressed at a low level on the plasma membrane and bind to sialic acid residues for immunosurveillance and cell-cell communication. Siglecs are classified based on their inhibitory and activatory downstream signaling properties. Inhibitory Siglecs negatively regulate microglia activation upon recognizing the intact sialic acid patterns and vice versa. MBLs are expressed upon infection in cytoplasm and can be secreted in order to recognize molecules containing terminal mannose as an innate immune

  19. Knowledge-based modeling of a legume lectin and docking of the carbohydrate ligand: the Ulex europaeus lectin I and its interaction with fucose.

    Science.gov (United States)

    Gohier, A; Espinosa, J F; Jimenez-Barbero, J; Carrupt, P A; Pérez, S; Imberty, A

    1996-12-01

    Ulex europaeus isolectin I is specific for fucose-containing oligosaccharide such as H type 2 trisaccharide alpha-L-Fuc (1-->2) beta-D-Gal (1-->4) beta-D-GlcNAc. Several legume lectins have been crystallized and modeled, but no structural data are available concerning such fucose-binding lectin. The three-dimensional structure of Ulex europaeus isolectin I has been constructed using seven legume lectins for which high-resolution crystal structures were available. Some conserved water molecules, as well as the structural cations, were taken into account for building the model. In the predicted binding site, the most probable locations of the secondary hydroxyl groups were determined using the GRID method. Several possible orientations could be determined for a fucose residue. All of the four possible conformations compatible with energy calculations display several hydrogen bonds with Asp-87 and Ser-132 and a stacking interaction with Tyr-220 and Phe-136. In two orientations, the O-3 and O-4 hydroxyl groups of fucose are the most buried ones, whereas two other, the O-2 and O-3 hydroxyl groups are at the bottom of the site. Possible docking modes are also studied by analysis of the hydrophobic and hydrophilic surfaces for both the ligand and the protein. The SCORE method allows for a quantitative evaluation of the complementarity of these surfaces, on the basis of molecular lipophilicity calculations. The predictions presented here are compared with known biochemical data.

  20. Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

  1. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    is not universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...

  2. Functional impact of HIV coreceptor-binding site mutations

    International Nuclear Information System (INIS)

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W.; Reeves, Jacqueline D.

    2006-01-01

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner

  3. Pet imaging of peripheral benzodiazepine binding sites in brain tumors

    International Nuclear Information System (INIS)

    Junck, L.; Jewett, D.M.; Olsen, J.M.; Kilbourn, M.R.; Koeppe, R.A.; Young, A.B.; Greenberg, H.S.; Kuhl, D.E.

    1991-01-01

    Studies in vitro have shown that the peripheral-type benzodiazepine binding site (PBBS) is present in moderate to high density on malignant gliomas as well as in areas of reactive gliosis, but in low density in normal brain. PK 11195 is an isoquinoline derivative that binds selectively to the PBBS but not to the central benzodiazepine receptor. We have used [ 11 C]PK 11195 with positron emission tomography (PET) to study brain tumors and cerebral infarcts. Preliminary results showed that, in 13 of 18 patients with astrocytomas, [ 11 C]PK 11195 radioactivity was increased in tumor compared to remote brain and that the concentration ratios of tumor-to-remote brain were higher for high grade astrocytomas than for low grade astrocytomas. Pharmacokinetic analysis suggests that the increased activity in tumor probably does not result from alterations in blood flow or vascular permeability. Patients with lymphoma, meningioma, medulloblastoma, brain metastasis, and neurosarcoidosis have also shown increased radioactivity in tumor. Among eight patients with acute and subacute cerebral infarcts, activity in the infarct was increased in seven and was often greatest at the periphery. We conclude that [ 11 C]PK 11195 is a promising radiopharmaceutical for further investigation of brain tumors as well as diseases characterized by reactive gliosis

  4. Muscarinic acetylcholine receptors: location of the ligand binding site

    International Nuclear Information System (INIS)

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-01-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

  5. Distance between two binding sites of the same antibody molecule

    International Nuclear Information System (INIS)

    Cser, L.; Gladkikh, I.A.; Ostanevich, Y.M.; Franek, F.; Novotny, J.; Nezlin, R.S.

    1978-01-01

    Neutron small-angle scattering experiments are reported, aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. Although the distance values could be determined only with a large statistical error, the data allowed the conclusion that the geometrical parameters of the complexes formed with the early (i.e., precipitating) antibody are significantly different from those of the complexes formed with the late (i.e, non-precipitating) antibody. The data suggest that the precipitating antibody complexed with a high molecular weight antigen assumes an extended shape with an antigen to antigen distance of 35.8 +- 1.3 nm. (Auth.)

  6. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  7. Purification and characterization of Cc-Lec, C-type lactose-binding lectin: A platelet aggregation and blood-clotting inhibitor from Cerastes cerastes venom.

    Science.gov (United States)

    Samah, Saoud; Fatah, Chérifi; Jean-Marc, Berjeaud; Safia, Kellou-Taîri; Fatima, Laraba-Djebari

    2017-09-01

    In this study, we reported for the first time the biochemical and structural characterization of Cc-Lec, a C-type lectin purified from Cerastes cerastes venom by affinity chromatography. This lectin was homogeneous by SDS-PAGE, and was shown to be a 34 271.59Da polypeptide by Electrospray mass spectrometry MS-ES-TOF. Its identified sequence of 160 amino acids corresponding to one subunit, revealed a high identity with other related proteins. Cc-Lec modeled 3D structure appeared as homodimer cross-linked by one disulfide bridge. Cc-Lec exhibited a calcium dependent hemagglutinating activity against human group O erythrocytes. Cc-Lec inhibited platelet aggregation induced by ADP, arachidonic acid or fibrinogen suggesting its interaction with their specific receptors namely P2Y1 and/or P2Y12, GPIIb/IIIa and TPα respectively. Cc-Lec was not lethal for mice until 10mg/kg administered by i.p. route. The lectin displayed a lasting anticoagulation on mice plasma even two days post-injection. This anticoagulation seems to be related to its interaction with coagulation factors Xa and IXa. Therefore, Cc-Lec prevented FXa amidolytic activity with Km=4.3310 -4 μg/mL and ki=14.4μg/mL. It seems to interact with these targets through CRD domain which could make it a good target as a pharmacological promising molecule in thrombosis diagnosis and therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

    DEFF Research Database (Denmark)

    Beatson, Richard; Maurstad, Gjertrud; Picco, Gianfranco

    2015-01-01

    Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through...... carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell...

  9. Target-mediated drug disposition model for drugs with two binding sites that bind to a target with one binding site.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2017-10-01

    The paper extended the TMDD model to drugs with two identical binding sites (2-1 TMDD). The quasi-steady-state (2-1 QSS), quasi-equilibrium (2-1 QE), irreversible binding (2-1 IB), and Michaelis-Menten (2-1 MM) approximations of the model were derived. Using simulations, the 2-1 QSS approximation was compared with the full 2-1 TMDD model. As expected and similarly to the standard TMDD for monoclonal antibodies (mAb), 2-1 QSS predictions were nearly identical to 2-1 TMDD predictions, except for times of fast changes following initiation of dosing, when equilibrium has not yet been reached. To illustrate properties of new equations and approximations, several variations of population PK data for mAbs with soluble (slow elimination of the complex) or membrane-bound (fast elimination of the complex) targets were simulated from a full 2-1 TMDD model and fitted to 2-1 TMDD models, to its approximations, and to the standard (1-1) QSS model. For a mAb with a soluble target, it was demonstrated that the 2-1 QSS model provided nearly identical description of the observed (simulated) free drug and total target concentrations, although there was some minor bias in predictions of unobserved free target concentrations. The standard QSS approximation also provided a good description of the observed data, but was not able to distinguish between free drug concentrations (with no target attached and both binding site free) and partially bound drug concentrations (with one of the binding sites occupied by the target). For a mAb with a membrane-bound target, the 2-1 MM approximation adequately described the data. The 2-1 QSS approximation converged 10 times faster than the full 2-1 TMDD, and its run time was comparable with the standard QSS model.

  10. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

    DEFF Research Database (Denmark)

    Long, Katherine S; Porse, Bo T

    2003-01-01

    , of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site...... on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome....

  11. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    International Nuclear Information System (INIS)

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  12. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

    Science.gov (United States)

    Clifford, Jacob; Adami, Christoph

    2015-09-02

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

  13. Osteopontin: A uranium phosphorylated binding-site characterization

    International Nuclear Information System (INIS)

    Safi, Samir; Jeanson, Aurelie; Roques, Jerome; Simoni, Eric; Creff, Gaelle; Qi, Lei; Basset, Christian; Vidaud, Claude; Solari, Pier Lorenzo; Den Auwer, Christophe

    2013-01-01

    Herein, we describe the structural investigation of one possible uranyl binding site inside a non structured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phospho-peptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U L(III)-edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO 2 2+ /peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein. (authors)

  14. Digestion and Interaction of Starches with α-Amylases: I. Mutational analysis of Carbohydrate Binding Sites in barley. II. In Vitro Starch Digestion of Legumes

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch

    2006-01-01

    the hydrolysis of internal 1,4-α-D-glucosidic bonds in starch and related polysaccharides. The present thesis concerns studies of two α-amylases: 1) secondary substrate binding sites in barley α-amylase 1 (AMY1), and 2) the involvement of anti-nutrients in in vitro digestion of starch in legumes by porcine...... in morphology between high amylose starch granules and normal starch granules. Legumes (beans, peas, and lentils) are characterised by low blood glucose raising potential, which is proportional to the in vitro starch digestion rates. The high amount of anti-nutritional factors (phytate, proteinaceous inhibitors......, tannins, and lectins) in legumes has been associated with the slow starch digestion. However, it is still debated in literature to which extent the legume starch digestibility is affected by anti-nutritional factors. The in vitro starch digestion (hydrolytic index, HI) of pea (Pisum sativum) and mixtures...

  15. Current status of lectin-based cancer diagnosis and therapy

    Directory of Open Access Journals (Sweden)

    Fohona S. Coulibaly

    2017-01-01

    Full Text Available Lectins are carbohydrate recognizing proteins originating from diverse origins in nature, including animals, plants, viruses, bacteria and fungus. Due to their exceptional glycan recognition property, they have found many applications in analytical chemistry, biotechnology and surface chemistry. This manuscript explores the current use of lectins for cancer diagnosis and therapy. Moreover, novel drug delivery strategies aiming at improving lectin’s stability, reducing their undesired toxicity and controlling their non-specific binding interactions are discussed. We also explore the nanotechnology application of lectins for cancer targeting and imaging. Although many investigations are being conducted in the field of lectinology, there is still a limited clinical translation of the major findings reported due to lectins stability and toxicity concerns. Therefore, new investigations of safe and effective drug delivery system strategies for lectins are warranted in order to take full advantage of these proteins.

  16. Prediction of nucleosome positioning based on transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Xianfu Yi

    Full Text Available BACKGROUND: The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs have been suggested to play a role in nucleosome positioning in vivo. PRINCIPAL FINDINGS: Here, the minimum redundancy maximum relevance (mRMR feature selection algorithm, the nearest neighbor algorithm (NNA, and the incremental feature selection (IFS method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test. CONCLUSIONS: Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.

  17. Binding of MCM-interacting proteins to ATP-binding site in MCM6

    Directory of Open Access Journals (Sweden)

    Hosoi A

    2016-03-01

    Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

  18. Lectin typing of Campylobacter concisus

    DEFF Research Database (Denmark)

    Aabenhus, Rune Munck; Hynes, Sean O; Permin, Henrik

    2002-01-01

    A total of 44 clinical isolates and the type strain of the putative pathogen Campylobacter concisus were grouped based on their reactions with plant lectins. The optimized lectin typing system used C. concisus strains proteolytically pretreated and subsequently typed by using a panel of four...... lectins. The system grouped all 45 strains into 13 lectin reaction patterns, leaving no strain untypeable due to autoagglutination. Lectin types were both stable and reproducible....

  19. Glycoprofiling of Early Gastric Cancer Using Lectin Microarray Technology.

    Science.gov (United States)

    Li, Taijie; Mo, Cuiju; Qin, Xue; Li, Shan; Liu, Yinkun; Liu, Zhiming

    2018-01-01

    Recently, studies have reported that protein glycosylation plays an important role in the occurrence and development of cancer. Gastric cancer is a common cancer with high morbidity and mortality owing to most gastric cancers are discovered only at an advanced stage. Here, we aim to discover novel specific serum glycanbased biomarkers for gastric cancer. A lectin microarray with 50 kinds of tumor-associated lectin was used to detect the glycan profiles of serum samples between early gastric cancer and healthy controls. Then lectin blot was performed to validate the differences. The result of the lectin microarray showed that the signal intensities of 13 lectins showed significant differences between the healthy controls and early gastric cancer. Compared to the healthy, the normalized fluorescent intensities of the lectins PWA, LEL, and STL were significantly increased, and it implied that their specifically recognized GlcNAc showed an especially elevated expression in early gastric cancer. Moreover, the binding affinity of the lectins EEL, RCA-II, RCA-I, VAL, DSA, PHA-L, UEA, and CAL were higher in the early gastric cancer than in healthy controls. These glycan structures containing GalNAc, terminal Galβ 1-4 GlcNAc, Tri/tetraantennary N-glycan, β-1, 6GlcNAc branching structure, α-linked fucose residues, and Tn antigen were elevated in gastric cancer. While the two lectins CFL GNL reduced their binding ability. In addition, their specifically recognized N-acetyl-D-galactosamine structure and (α-1,3) mannose residues were decreased in early gastric cancer. Furthermore, lectin blot results of LEL, STL, PHA-L, RCA-I were consistent with the results of the lectin microarray. The findings of our study clarify the specific alterations for glycosylation during the pathogenesis of gastric cancer. The specific high expression of GlcNAc structure may act as a potential early diagnostic marker for gastric cancer.

  20. Lectin activity in mycelial extracts of Fusarium species.

    Science.gov (United States)

    Bhari, Ranjeeta; Kaur, Bhawanpreet; Singh, Ram S

    2016-01-01

    Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycoproteins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, whereas the protease treatment resulted in a significant decline in agglutination by most of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of Fusarium lectins exhibited binding affinity to d-ribose, l-fucose, d-glucose, l-arabinose, d-mannitol, d-galactosamine hydrochloride, d-galacturonic acid, N-acetyl-d-galactosamine, N-acetyl-neuraminic acid, 2-deoxy-d-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Lectins in fish skin: do they play a role in host-monogenean interactions?

    Science.gov (United States)

    Buchmann, K

    2001-09-01

    Mucus samples from rainbow trout skin with or without infections by Gyrodactylus derjavini were tested for the presence of lectins reacting with mannose, galactose and lactose. The samples inhibited the binding of biotinylated lectins (from Canavalia ensiformis, Artocarpus integrifolia and Erythrina corallodendron, respectively) to microtitre plates with covalently bound carbohydrates (mannopyranoside, galactopyranoside and lactose, respectively). However, the inhibition of C. ensiformis and A. integrifolia lectins was slightly greater when mucus from infected (but recovering) fish was used, suggesting an increase of mannose and galactose binding lectins in fish skin exposed to parasites. As mannose, galactose and lactose are present on the glycocalyx of Gyrodactylus derjavini, it is suggested that lectins could play a dual role in interactions between fish hosts and their monogenean parasites. Thus, recognition between parasite and host and also host responses towards parasite infections could both, at least partly, involve carbohydrate-lectin binding.

  2. Serine protease immunohistochemistry and lectin histochemistry in the small intestine of weaned and unweaned pigs

    DEFF Research Database (Denmark)

    Brown, P J; Poulsen, Steen Seier; Wells, M

    1991-01-01

    The distribution of goblet cells containing serine protease and of those binding the lectin Ulex europaeus agglutinin-1 (UEA-1) in the pig small intestine is altered during the period after weaning. Goblet cells exhibiting binding of other lectins were not altered. These alterations and other...

  3. Functional and physical molecular size of the chicken hepatic lectin determined by radiation inactivation and sedimentation equilibrium analysis

    International Nuclear Information System (INIS)

    Steer, C.J.; Osborne, J.C. Jr.; Kempner, E.S.

    1990-01-01

    Radiation inactivation and sedimentation equilibrium analysis were used to determine the functional and physical size of the chicken hepatic membrane receptor that binds N-acetylglucosamine-terminated glycoproteins. Purified plasma membranes from chicken liver were irradiated with high energy electrons and assayed for 125I-agalactoorosomucoid binding. Increasing the dose of ionizing radiation resulted in a monoexponential decay in binding activity due to a progressive loss of binding sites. The molecular mass of the chicken lectin, determined in situ by target analysis, was 69,000 +/- 9,000 Da. When the same irradiated membranes were solubilized in Brij 58 and assayed, the binding protein exhibited a target size of 62,000 +/- 4,000 Da; in Triton X-100, the functional size of the receptor was 85,000 +/- 10,000 Da. Sedimentation equilibrium measurements of the purified binding protein yielded a lower limit molecular weight of 79,000 +/- 7,000. However, the solubilized lectin was detected as a heterogeneous population of oligomers with molecular weights as high as 450,000. Addition of calcium or calcium plus N-acetylglucosamine decreased the higher molecular weight species, but the lower limit molecular weights remained invariant. Similar results were determined when the chicken lectin was solubilized in Brij 58, C12E9, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). Results from the present study suggest that in the plasma membrane, the functional species of the chicken hepatic lectin exists as a trimer. However, in detergent solution, the purified receptor forms a heterogeneous population of irreversible oligomers that exhibit binding activity proportional to size

  4. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  5. In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.

    Directory of Open Access Journals (Sweden)

    Nima Najand

    Full Text Available We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.

  6. Lectin histochemical evaluation of glycoconjugates in dog efferent ductules.

    Science.gov (United States)

    Wakui, S; Furusato, M; Takahashi, H; Motoya, M; Ushigome, S

    1996-06-01

    Glycoconjugates in the epithelial cells of the efferent ductules in the dog were investigated using lectin histochemistry. These ductules connect the extratesticular rete with the epididymis. The epithelium of the ductules consisted both of ciliated and nonciliated cells. Whereas the apical zone of ciliated cells showed selective binding with WGA, SWGA, SNA, MAA and neuraminidase-PNA, that of nonciliated cells bound to all lectins used in the present study: WGA, SWGA, SNA, MAA, PNA, neuraminidase-PNA, RCA1, DBA and SBA. The nonciliated cells were divided into 3 types: type A cells which lacked both specific granules and vacuoles, type B cells which were characterised by a few specific apical vacuoles and many large specific granules, and type C cells which were characterised by some specific apical vacuoles and small basal granules. The specific granules and vacuoles of type B cells showed binding with WGA, SWGA and MAA. The specific granules of type C cells showed binding with WGA, SWGA, SNA, MAA, PNA and neuraminidase-PNA, while their specific vacuoles showed binding with WGA, SWGA, SNA and MAA. The Golgi zone both of ciliated and type A cells did not bind with any lectins used in this study, while type B and C cells showed similar lectin binding patterns between the Golgi zone and their specific granules. Specific lectin binding patterns revealed a different carbohydrate composition of each type of cell, indicating a biological difference between the ciliated cells and the 3 types of nonciliated cells in dog efferent ductules.

  7. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    Energy Technology Data Exchange (ETDEWEB)

    Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

    2013-03-15

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

  8. Fungal lectin MpL enables entry of protein drugs into cancer cells and their subcellular targeting

    OpenAIRE

    ?urga, Simon; Nanut, Milica Peri?i?; Kos, Janko; Saboti?, Jerica

    2017-01-01

    Lectins have been recognized as promising carrier molecules for targeted drug delivery. They specifically bind carbohydrate moieties on cell membranes and trigger cell internalization. Fungal lectin MpL (Macrolepiota procera lectin) does not provoke cancer cell cytotoxicity but is able to bind aminopeptidase N (CD13) and integrin ?3?1, two glycoproteins that are overexpressed on the membrane of tumor cells. Upon binding, MpL is endocytosed in a clathrin-dependent manner and accumulates initia...

  9. Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Amy L Bauer

    2010-11-01

    Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

  10. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  11. Insights into Animal and Plant Lectins with Antimicrobial Activities

    Directory of Open Access Journals (Sweden)

    Renata de Oliveira Dias

    2015-01-01

    Full Text Available Lectins are multivalent proteins with the ability to recognize and bind diverse carbohydrate structures. The glyco -binding and diverse molecular structures observed in these protein classes make them a large and heterogeneous group with a wide range of biological activities in microorganisms, animals and plants. Lectins from plants and animals are commonly used in direct defense against pathogens and in immune regulation. This review focuses on sources of animal and plant lectins, describing their functional classification and tridimensional structures, relating these properties with biotechnological purposes, including antimicrobial activities. In summary, this work focuses on structural-functional elucidation of diverse lectin groups, shedding some light on host-pathogen interactions; it also examines their emergence as biotechnological tools through gene manipulation and development of new drugs.

  12. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    International Nuclear Information System (INIS)

    James, I.F.; Goldstein, A.

    1984-01-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  13. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  14. Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

    International Nuclear Information System (INIS)

    Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.; Goberna, R.; Guerrero, J.M.

    1991-01-01

    The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using [ 125 I]melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37 degree C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of [ 125 I]melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of [ 125 I]melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the [ 125 I]melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland

  15. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    Directory of Open Access Journals (Sweden)

    Chih-Hao Lu

    2015-01-01

    Full Text Available We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

  16. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  17. 8-anilino-1-naphthaline sulfonate binds at the hemoglobin allosteric regulatory sites: inhibitory analyses

    International Nuclear Information System (INIS)

    Bokut', S.B.; Parul', D.A.; Yachnik, N.N.; Milyutin, A.A.

    2001-01-01

    The present study focused on the localization at least one of the ANS binding sites in the major form of human hemoglobin HbA. High-resolution docking predict ANS binding to the hemoglobin central cavity. Steady-state fluorescence titration data obtained in the absence/presence of natural effector inositol hexaphosphate (IHP) allowed to conclude that IHP competitively inhibited ANS binding to HbA. Thus, we must conclude that one of the ANS binding sites is central cavity, which makes it possible to monitor changes at this region upon ligation/deligation, effector binding and changes in hemoglobin structure

  18. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    Science.gov (United States)

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Evidence for a non-opioid sigma binding site din the guinea-pig myenteric plexus

    International Nuclear Information System (INIS)

    Roman, F.; Pascaud, X.; Vauche, D.; Junien, J.

    1988-01-01

    The presence of a binding site to (+)-( 3 H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site and a low affinity site. Morphine and naloxone 10 -4 M were unable to displace (+)-( 3 H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig

  20. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    Science.gov (United States)

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

  1. Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

    Science.gov (United States)

    Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

    2017-10-01

    While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle

    DEFF Research Database (Denmark)

    Kjeldsen, K; Richter, Erik; Galbo, H

    1986-01-01

    ]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0...

  3. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...

  4. GABAA [gamma-aminobutyric acid] type binding sites on membranes of spermatozoa

    International Nuclear Information System (INIS)

    Erdoe, S.L.; Wekerle, L.

    1990-01-01

    The binding of [ 3 H] gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABA A type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa

  5. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    Science.gov (United States)

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

    Directory of Open Access Journals (Sweden)

    Magdalena eEriksson

    2013-07-01

    Full Text Available Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern recognition receptors sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs are pattern recognition receptors recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR SIGNR3 in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homologue of the human DC-SIGN receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze if this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium (DSS-induced colitis model was employed. SIGNR3-/- mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3-/- mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

  7. Sugar residues content and distribution in atrophic and hyperplastic postmenopausal human endometrium: lectin histochemistry.

    Science.gov (United States)

    Gheri, G; Bryk, S G; Taddei, G; Moncini, D; Noci, I

    1996-10-01

    A lectin histochemical study was performed to investigate the glycoconjugate saccharidic moieties of the human postmenopausal endometrium (14 atrophic and 15 hyperplastic). For this purpose a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, ConA, LTA and UEA I) was used. No differences in lectin binding between atrophic and hyperplastic endometria were observed. This investigation allowed us to provide a basic picture of the oligosaccharidic distribution in postmenopausal endometria. The data on the saccharidic distribution at the postmenopausal endometria showed a large amount of sugar residues at all the investigated sites, i.e. the lining and glandular epithelium, the stroma and the vessels (capillary and large vessels). Furthermore, at the endometrial lining epithelium, at the glands and at the wall of the blood vessels of some postmenopausal women the presence of alpha-L-fucosyl residues which bind via alpha (1-6) linkage to penultimate glucosaminyl residues and/or difucosylated oligosaccharides was demonstrated for the first time.

  8. Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

    DEFF Research Database (Denmark)

    Nielbo, Steen Günther; Thomsen, J.K.; Graversen, J. H.

    2004-01-01

    . A conserved proline, which was found to be in the cis conformation in holoTN3, is in apoTN3 predominantly in the trans conformation. Backbone dynamics indicate that, in apoTN3 especially, two of the three calcium-binding loops and two of the three K4-binding residues exhibit increased flexibility, whereas...

  9. Mcm1p binding sites in ARG1 positively regulate Gcn4p binding and SWI/SNF recruitment

    OpenAIRE

    Yoon, Sungpil; Hinnebusch, Alan G.

    2009-01-01

    Transcription of the arginine biosynthetic gene ARG1 is activated by Gcn4p, a transcription factor induced by starvation for any amino acid. Previously we showed that Gcn4p binding stimulates the recruitment of Mcm1p and co-activator SWI/SNF to ARG1 in cells via Gcn4p induction through amino acid starvation. Here we report that Gcn4p binding is reduced by point mutations of the Mcm1p binding site and increased by overexpression of Mcm1p. This result suggests that Mcm1p plays a positive role i...

  10. ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin ulex europaeus agglutinin I.

    Science.gov (United States)

    Engelmann, B

    1993-11-01

    The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.

  11. The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

    Science.gov (United States)

    Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

    2017-12-05

    Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Sugar residues content and distribution in atrophic and hyperplastic postmenopausal human endometrium: lectin histochemistry

    OpenAIRE

    Gheri, G.; Gheri Bryk, S.; Taddei, G.; Moncini, D.; Noci, I.

    1996-01-01

    A lectin histochemical study was performed to investigate the glycoconjugate saccharidic moieties of the human postmenopausal endometrium (14 atrophic and 15 hyperplastic). For this purpose a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, ConA, LTA and UEA I) was used. No differences in lectin binding between atrophic and hyperplastic endometria were observed. This investigation allowed us to provide a basic picture of the oligo...

  13. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Lectins with Anti-HIV Activity: A Review

    Directory of Open Access Journals (Sweden)

    Ouafae Akkouh

    2015-01-01

    Full Text Available Lectins including flowering plant lectins, algal lectins, cyanobacterial lectins, actinomycete lectin, worm lectins, and the nonpeptidic lectin mimics pradimicins and benanomicins, exhibit anti-HIV activity. The anti-HIV plant lectins include Artocarpus heterophyllus (jacalin lectin, concanavalin A, Galanthus nivalis (snowdrop agglutinin-related lectins, Musa acuminata (banana lectin, Myrianthus holstii lectin, Narcissus pseudonarcissus lectin, and Urtica diocia agglutinin. The anti-HIV algal lectins comprise Boodlea coacta lectin, Griffithsin, Oscillatoria agardhii agglutinin. The anti-HIV cyanobacterial lectins are cyanovirin-N, scytovirin, Microcystis viridis lectin, and microvirin. Actinohivin is an anti-HIV actinomycete lectin. The anti-HIV worm lectins include Chaetopterus variopedatus polychaete marine worm lectin, Serpula vermicularis sea worm lectin, and C-type lectin Mermaid from nematode (Laxus oneistus. The anti-HIV nonpeptidic lectin mimics comprise pradimicins and benanomicins. Their anti-HIV mechanisms are discussed.

  15. Characterization of [3H] oxymorphone binding sites in mouse brain

    DEFF Research Database (Denmark)

    Yoo, Ji Hoon; Borsodi, Anna; Tóth, Géza

    2017-01-01

    Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear....... This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [3H]oxymorphone revealed high affinity binding sites in mouse......]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [3H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP...

  16. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  17. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  18. Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

    Science.gov (United States)

    Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

    2014-01-01

    Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The

  19. Glycans: bioactive signals decoded by lectins.

    Science.gov (United States)

    Gabius, Hans-Joachim

    2008-12-01

    The glycan part of cellular glycoconjugates affords a versatile means to build biochemical signals. These oligosaccharides have an exceptional talent in this respect. They surpass any other class of biomolecule in coding capacity within an oligomer (code word). Four structural factors account for this property: the potential for variability of linkage points, anomeric position and ring size as well as the aptitude for branching (first and second dimensions of the sugar code). Specific intermolecular recognition is favoured by abundant potential for hydrogen/co-ordination bonds and for C-H/pi-interactions. Fittingly, an array of protein folds has developed in evolution with the ability to select certain glycans from the natural diversity. The thermodynamics of this reaction profits from the occurrence of these ligands in only a few energetically favoured conformers, comparing favourably with highly flexible peptides (third dimension of the sugar code). Sequence, shape and local aspects of glycan presentation (e.g. multivalency) are key factors to regulate the avidity of lectin binding. At the level of cells, distinct glycan determinants, a result of enzymatic synthesis and dynamic remodelling, are being defined as biomarkers. Their presence gains a functional perspective by co-regulation of the cognate lectin as effector, for example in growth regulation. The way to tie sugar signal and lectin together is illustrated herein for two tumour model systems. In this sense, orchestration of glycan and lectin expression is an efficient means, with far-reaching relevance, to exploit the coding potential of oligosaccharides physiologically and medically.

  20. Animal lectins: potential receptors for ginseng polysaccharides

    Directory of Open Access Journals (Sweden)

    So Hee Loh

    2017-01-01

    Full Text Available Panax ginseng Meyer, belonging to the genus Panax of the family Araliaceae, is known for its human immune system-related effects, such as immune-boosting effects. Ginseng polysaccharides (GPs are the responsible ingredient of ginseng in immunomodulation, and are classified as acidic and neutral GPs. Although GPs participate in various immune reactions including the stimulation of immune cells and production of cytokines, the precise function of GPs together with its potential receptor(s and their signal transduction pathways have remained largely unknown. Animal lectins are carbohydrate-binding proteins that are highly specific for sugar moieties. Among many different biological functions in vivo, animal lectins especially play important roles in the immune system by recognizing carbohydrates that are found exclusively on pathogens or that are inaccessible on host cells. This review summarizes the immunological activities of GPs and the diverse roles of animal lectins in the immune system, suggesting the possibility of animal lectins as the potential receptor candidates of GPs and giving insights into the development of GPs as therapeutic biomaterials for many immunological diseases.

  1. Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

    International Nuclear Information System (INIS)

    Leslie, R.A.; McDonald, T.J.; Robertson, H.A.

    1988-01-01

    Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared [ 125 I]PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of [ 125 I]PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of [ 125 I]PYY binding sites throughout the rat brain was seen to be similar to the distribution of [ 125 I]NPY binding sites

  2. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

    Science.gov (United States)

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

    2011-05-31

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  3. Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

    Science.gov (United States)

    Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

    2011-05-01

    Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Druggable pockets and binding site centric chemical space: a paradigm shift in drug discovery.

    Science.gov (United States)

    Pérot, Stéphanie; Sperandio, Olivier; Miteva, Maria A; Camproux, Anne-Claude; Villoutreix, Bruno O

    2010-08-01

    Detection, comparison and analyses of binding pockets are pivotal to structure-based drug design endeavors, from hit identification, screening of exosites and de-orphanization of protein functions to the anticipation of specific and non-specific binding to off- and anti-targets. Here, we analyze protein-ligand complexes and discuss methods that assist binding site identification, prediction of druggability and binding site comparison. The full potential of pockets is yet to be harnessed, and we envision that better understanding of the pocket space will have far-reaching implications in the field of drug discovery, such as the design of pocket-specific compound libraries and scoring functions.

  5. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  6. Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

    Science.gov (United States)

    Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2013-07-01

    Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

  7. An overview of the prediction of protein DNA-binding sites.

    Science.gov (United States)

    Si, Jingna; Zhao, Rui; Wu, Rongling

    2015-03-06

    Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  8. Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins.

    Science.gov (United States)

    Lotan, R; Beattie, G; Hubbell, W; Nicolson, G L

    1977-05-03

    The effects of several commonly used detergents on the saccharide-binding activities of lectins were investigated using lectin-mediated agglutination of formalin-fixed erythrocytes and affinity chromatography of glycoproteins on columns of lectins immobilized on polyacrylic hydrazide-Sepharose. In the hemagglutination assays, Ricinus communis I (RCA1) and II (RCAII), concanavalin A (Con A), and the agglutinins from peanut (PNA), soybean (SBA), wheat germ (WGA), and Limulus polyphemus (LPA) were tested with several concentrations of switterionic, cationic, anionic, and nonionic detergents. It was found that increasing detergent concentrations eventually affected hemagglutination titers in both test and control samples, and the highest detergent concentrations not affecting lectin hemagglutinating activities were determined. The effects of detergents on specific binding of [3H]fetuin and asialo[3H]fetuin to and elution from columns of immobilized lectins were less severe when compared with lectins in solution, suggesting that the lectins are stabilized by covalent attachment to agarose beads. Nonionic detergents did not affect the binding efficiency of the immobilized lectins tested at concentrations used for membrane solubilization while cationic and zwitterionic detergents caused significant inhibition of Con A- and SBA-Sepharose activities. In sodium deoxycholate (greater than 1%) only RCAI-Sepharose retained its activity, whereas the activities of the other lectins were reduced dramatically. Low concentrations of sodium dodecyl sulfate (0.05%) inhibited only the activity of immobilized SBA, but at higher concentration (0.1%) and prolonged periods of incubation (16 h, 23 degrees C) most of the lectins were inactivated. These data are compared with previous reports on the use of detergents in lectin affinity chromatography, and the conditions for the optimal use of detergents are detailed.

  9. Location and nature of calcium-binding sites in salivary acidic proline-rich phosphoproteins

    International Nuclear Information System (INIS)

    Bennick, A.; McLaughlin, A.C.; Grey, A.A.; Madapallimattam, G.

    1981-01-01

    The location of the calcium-binding sites in the human acidic proline-rich proteins, salivary proteins A and C, was determined by equilibrium dialysis of the tryptic peptides with buffers containing 45 Ca. All the calcium-binding sites are located in the NH 2 -terminal tryptic peptide (TX peptide). The nature of the calcium binding sites in the TX peptide and native salivary proteins A and C, as well as dephosphorylated proteins was compared. Two types of sites can be distinguished in peptide TX. Type I sites have an apparent dissociation constant (K) of 38 μM and are responsible for the binding of 2.6 mol of Ca/mol of peptide. The corresponding figures for Type II sites are 780 μM and 5.3 mol of Ca/mol of peptide. In the native proteins, the amount of calcium bound at the type II sites decreases to 3.9 mol of Ca/mol of proteins A and C and K increases to 1100 μM. The amount of calcium bound at type I sites decreases to 1.5 mol/mol of protein A and 0.6 mol/mol of protein C, but there is no change in K. Dephosphorylation affects the calcium binding at both types of sites. The experiments indicate that the COOH-terminal parts of the native proteins affect the number and the nature of the protein calcium-binding sites. Proton and phosphorous NMR data demonstrate that β-COOH in aspartic acid, as well as phosphoserine, are part of the calcium-binding sites. The difference in calcium binding to salivary proteins A and C may be due at least partially to differences in the environment of one or more aspartic acids

  10. Genome-wide identification of estrogen receptor alpha-binding sites in mouse liver

    DEFF Research Database (Denmark)

    Gao, Hui; Fält, Susann; Sandelin, Albin

    2007-01-01

    We report the genome-wide identification of estrogen receptor alpha (ERalpha)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERalpha-binding regions...... genes. The majority of ERalpha-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS...... signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERalpha to DNA in intact chromatin....

  11. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

    Directory of Open Access Journals (Sweden)

    Oscar Harari

    2010-07-01

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  12. Ligand-binding sites in human serum amyloid P component

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.

    1996-01-01

    Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes, AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly...

  13. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  14. Human Lectins and Their Roles in Viral Infections

    Directory of Open Access Journals (Sweden)

    Christopher P. Mason

    2015-01-01

    Full Text Available Innate recognition of virus proteins is an important component of the immune response to viral pathogens. A component of this immune recognition is the family of lectins; pattern recognition receptors (PRRs that recognise viral pathogen-associated molecular patterns (PAMPs including viral glycoproteins. In this review we discuss the contribution of soluble and membrane-associated PRRs to immunity against virus pathogens, and the potential role of these molecules in facilitating virus replication. These processes are illustrated with examples of viruses including human immunodeficiency virus (HIV, hepatitis C virus (HCV and Ebola virus (EBOV. We focus on the structure, function and genetics of the well-characterised C-type lectin mannose-binding lectin, the ficolins, and the membrane-bound CD209 proteins expressed on dendritic cells. The potential for lectin-based antiviral therapies is also discussed.

  15. Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

    Science.gov (United States)

    Horejsí, V; Tichá, M; Kocourek, J

    1977-09-29

    Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

  16. Impact of germline and somatic missense variations on drug binding sites.

    Science.gov (United States)

    Yan, C; Pattabiraman, N; Goecks, J; Lam, P; Nayak, A; Pan, Y; Torcivia-Rodriguez, J; Voskanian, A; Wan, Q; Mazumder, R

    2017-03-01

    Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein's gene. To identify the nsSNVs that may affect drug binding, protein-drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein-drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein-drug binding sites. Using this method we identified 12 993 amino acid-drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid-drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid-drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid-drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein-drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein-drug binding predicted drug target proteins and prevalence of both somatic and

  17. Lectin histochemistry of 1,2-dimethylhydrazine-induced rat colon neoplasia.

    Science.gov (United States)

    Freeman, H J

    1983-10-01

    Lectins linked to fluorescein were used as carbohydrate probes to examine the goblet cell mucin and epithelial cell surface glycoconjugate alterations in an experimental rodent model of colonic neoplasia induced with parenteral 1,2-dimethylhydrazine dihydrochloride. Lectins derived from Triticum vulgare (WGA), Ricinus communis (RCA1), and Limulus polyphemus (LPA) showed reduced labeling of goblet cell mucin in these tumors, while binding with peanut lectin from Arachis hypogaea (PNA), a lectin ordinarily failing to bind to mucin in normal colon, was positive. In addition, RCA1 and LPA showed increased cell surface labeling of neoplastic epithelial cells. Finally, alterations were observed in lectin binding to "transitional" colonic mucosa adjacent to colonic tumors from carcinogen-treated rats. These findings indicate that significant alterations in both membrane and mucin glycoconjugates occur in colonic tumors and mucosa adjacent to tumors in a chemically induced experimental animal model of human colon cancer.

  18. Quantitative autoradiographic distribution of L-[3H]glutamate-binding sites in rat central nervous system

    International Nuclear Information System (INIS)

    Greenamyre, J.T.; Young, A.B.; Penney, J.B.

    1984-01-01

    Quantitative autoradiography was used to determine the distribution of L-[3H]glutamate-binding sites in the rat central nervous system. Autoradiography was carried out in the presence of Cl- and Ca2+ ions. Scatchard plots and Hill coefficients of glutamate binding suggested that glutamate was interacting with a single population of sites having a K-D of about 300 nM and a capacity of 14.5 pmol/mg of protein. In displacement studies, ibotenate also appeared to bind to a single class of non-interacting sites with a KI of 28 microM. However, quisqualate displacement of [3H]glutamate binding revealed two well-resolved sites with KIS of 12 nM and 114 microM in striatum. These sites were unevenly distributed, representing different proportions of specific glutamate binding in different brain regions. The distribution of glutamate-binding sites correlated very well with the projection areas of putative glutamatergic pathways. This technique provides an extremely sensitive assay which can be used to gather detailed pharmacological and anatomical information about L-[3H]glutamate binding in the central nervous system

  19. Quantitative autoradiography of [3H]ouabain binding sites in rat brain

    International Nuclear Information System (INIS)

    Spyropoulos, A.C.; Rainbow, T.C.

    1984-01-01

    In vitro quantitative autoradiography was used to localize in rat brain binding sites for [ 3 H]ouabain, an inhibitor of the Na + ,K + -ATPase. High levels of [ 3 H]ouabain sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [ 3 H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [ 14 C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [ 3 H]ouabain binding, indicating that some of the [ 3 H]ouabain binding sites are localized to neurons. The close correlation between [ 3 H]ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na + ,K + -ATPase. (Auth.)

  20. Leveraging cross-species transcription factor binding site patterns

    DEFF Research Database (Denmark)

    Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward

    2014-01-01

    Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to...... that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms....

  1. Photoaffinity studies of the tubulin-colchicine binding site

    International Nuclear Information System (INIS)

    Hahn, K.M.

    1987-01-01

    A variety of colchicine derivatives were synthesized and coupled with 3,3,3-trifluoro-2-diazapropionyl chloride (TFDP-Cl) to produce colchicine photoaffinity analogs for use in tubulin labelling studies. Photoaffinity analogs of allocolchicine and podophylotoxin were also made using the same photoreactive moiety. Several labels were found to be effective inhibitors of tubulin polymerization. The approximate tubulin binding constants of the labels, calculated from polymerization inhibition data, varied between 2.2 x 10 5 to 2.5 x 10 3 M -1 . The labels chosen for use in tubulin labelling experiments were (N-TFDP) deacetyl-thiocolchicine 1, (O-TFDP)thiocolchifoline 2, and (O-TFDP)-2-demethylthiocolchicine 3. Compound 1 was found to bind tubulin reversibly and to competitively inhibit colchicine binding. Methods for the incorporation of tritium and 14 C in these labels were developed. Conditions were found which caused labels to insert into solvent without photorearrangement of the colchicine skeleton. Catalytic base caused the α-diazo amide of 1 to rearrange to a triazole

  2. Europium ion as a probe for binding sites to carrageenans

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, Ana P.; Goncalves, Rogeria R.; Serra, Osvaldo A. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil); Zaniquelli, Maria Elisabete D. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil)], E-mail: medzaniquelli@ffclrp.usp.br; Wong, Kenneth [Laboratorio de Fisico-Quimica, Centro de Pesquisas de Paulinia, Rhodia Brasil, Paulinia, Sao Paulo (Brazil)

    2007-12-15

    Carrageenans, sulfated polysaccharides extracted from red algae, present a coil-helix transition and helix aggregation dependence on the type and concentration of counterions. In this study, we focus attention on a mixed valence counterion system: Eu{sup 3+}/Na{sup +} or K{sup +} with different gel-forming carrageenans: kappa, iota, and kappa-2. Results of stationary and time-dependent luminescence showed to be a suitable tool to probe ion binding to both the negatively charged sulfate group and the hydroxyl groups present in the biopolymer. For lower europium ion concentrations, a single longer decay emission lifetime was detected, which was attributed to the binding of europium ion to the carrageenan sulfate groups. An additional decay ascribed to europium binding to hydroxyl groups was observed above a threshold concentration, and this decay was dependent on the carrageenan charge density. Symmetry of the europium ion microenvironment was estimated by the ratio between the intensities of its emission bands, which has been shown to depend on the concentration of europium ions and on the specificity of the monovalent counterion bound to the carrageenan.

  3. Europium ion as a probe for binding sites to carrageenans

    International Nuclear Information System (INIS)

    Ramos, Ana P.; Goncalves, Rogeria R.; Serra, Osvaldo A.; Zaniquelli, Maria Elisabete D.; Wong, Kenneth

    2007-01-01

    Carrageenans, sulfated polysaccharides extracted from red algae, present a coil-helix transition and helix aggregation dependence on the type and concentration of counterions. In this study, we focus attention on a mixed valence counterion system: Eu 3+ /Na + or K + with different gel-forming carrageenans: kappa, iota, and kappa-2. Results of stationary and time-dependent luminescence showed to be a suitable tool to probe ion binding to both the negatively charged sulfate group and the hydroxyl groups present in the biopolymer. For lower europium ion concentrations, a single longer decay emission lifetime was detected, which was attributed to the binding of europium ion to the carrageenan sulfate groups. An additional decay ascribed to europium binding to hydroxyl groups was observed above a threshold concentration, and this decay was dependent on the carrageenan charge density. Symmetry of the europium ion microenvironment was estimated by the ratio between the intensities of its emission bands, which has been shown to depend on the concentration of europium ions and on the specificity of the monovalent counterion bound to the carrageenan

  4. The Lectin Complement Pathway in Patients with Necrotizing Soft Tissue Infection

    DEFF Research Database (Denmark)

    Hansen, Marco Bo; Rasmussen, Lars S; Pilely, Katrine

    2016-01-01

    BACKGROUND: Mannose-binding lectin (MBL) and ficolins are pattern recognition molecules (PRMs) that play an important role during infection through activation of the lectin complement pathway. We assessed whether plasma PRM levels were associated with mortality in patients with necrotizing soft t...

  5. C-type lectins do not act as functional receptors for filovirus entry into cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuno, Keita; Nakayama, Eri; Noyori, Osamu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan); Marzi, Andrea; Ebihara, Hideki [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Irimura, Tatsuro [Graduate School of Pharmaceutical Science, University of Tokyo, Tokyo (Japan); Feldmann, Heinz [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Takada, Ayato, E-mail: atakada@czc.hokudai.ac.jp [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan)

    2010-12-03

    Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

  6. C-type lectins do not act as functional receptors for filovirus entry into cells

    International Nuclear Information System (INIS)

    Matsuno, Keita; Nakayama, Eri; Noyori, Osamu; Marzi, Andrea; Ebihara, Hideki; Irimura, Tatsuro; Feldmann, Heinz; Takada, Ayato

    2010-01-01

    Research highlights: → Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. → Mutant GPs mediated virus entry less efficiently than wild-type GP. → Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. → C-type lectins do not independently mediate filovirus entry into cells. → Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

  7. Localization of 125I-insulin binding sites in the rat hypothalamus by quantitative autoradiography

    International Nuclear Information System (INIS)

    Corp, E.S.; Woods, S.C.; Figlewicz, D.P.; Porte, D. Jr.; Baskin, D.G.; Dorsa, D.M.

    1986-01-01

    In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125 I-insulin in the hypothalamus of the rat brain. Highest specific binding was found in the arculate, dorsomedial, suprachiasmatic, paraventricular and periventricular regions. Significantly lower binding was present in the ventromedial nucleus and median eminence. The results are consistent with the hypothesis that insulin modulates the neural regulation of feeding by acting at sites in the hypothalamus. (author)

  8. Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

    Energy Technology Data Exchange (ETDEWEB)

    Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

    1988-09-01

    Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples.

  9. Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

    International Nuclear Information System (INIS)

    Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

    1988-01-01

    Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples

  10. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

  11. Polar bear hemoglobin and human Hb A0: same 2,3-diphosphoglycerate binding site but asymmetry of the binding?

    Science.gov (United States)

    Pomponi, Massimo; Bertonati, Claudia; Patamia, Maria; Marta, Maurizio; Derocher, Andrew E; Lydersen, Christian; Kovacs, Kit M; Wiig, Oystein; Bårdgard, Astrid J

    2002-11-01

    Polar bear (Ursus maritimus) hemoglobin (Hb) shows a low response to 2,3-diphosphoglycerate (2,3-DPG), compared to human Hb A0, even though these proteins have the same 2,3-DPG-binding site. In addition, polar bear Hb shows a high response to chloride and an alkaline Bohr effect (deltalog P50/deltapH) that is significantly greater than that of human Hb A0. The difference in sequence Pro (Hb A0)-->Gly (polar bear Hb) at position A2 in the A helix seems to be critical for reduced binding of 2,3-DPG. Our results also show that the A2 position may influence not only the flexibility of the A helix, but that differences in flexibility of the first turn of the A helix may affect the unloading of oxygen for the intrinsic ligand affinities of the alpha and beta chains. However, preferential binding to either chain can only take place if there is appreciable asymmetric binding of the phosphoric effector. Regarding this point, 31P NMR data suggest a loss of symmetry of the 2,3-DPG-binding site in the deoxyHb-2,3-DPG complex.

  12. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  13. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  14. Exploring the composition of protein-ligand binding sites on a large scale.

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

    Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

  15. Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2018-02-01

    The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

  16. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  17. DNA Binding Drugs Targeting the Regulatory DNA Binding Site of the ETS Domain Family Transcription Factor Associated With Human Breast Cancer

    National Research Council Canada - National Science Library

    Wang, Yong-Dong

    1999-01-01

    .... The key approach is to prevent the binding of two transcription factors, ESX and AP-2, to the consensus DNA binding sites contained within the Her2/neu promoter resulting in inhibition of transcription factor function...

  18. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  19. Identification of an allosteric binding site for RORγt inhibition

    NARCIS (Netherlands)

    Scheepstra, M.; Leysen, S.; van Almen, G.; Miller, J.R.; Piesvaux, J.; Kutilek, V.; van Eenennaam, H.; Zhang, H.; Barr, K.; Nagpal, S.; Soisson, S.M.; Kornienko, M.; Wiley, K.; Elsen, N.; Sharma, S.; Correll, C.C.; Trotter, B.W.; Stelt, van der M.; Oubrie, A.; Ottmann, C.; Parthasarathy, G.; Brunsveld, L.

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of

  20. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  1. Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome.

    Science.gov (United States)

    Dresch, Jacqueline M; Zellers, Rowan G; Bork, Daniel K; Drewell, Robert A

    2016-01-01

    A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.

  2. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  3. CONREAL web server: identification and visualization of conserved transcription factor binding sites

    NARCIS (Netherlands)

    Berezikov, E.; Guryev, V.; Cuppen, E.

    2005-01-01

    The use of orthologous sequences and phylogenetic footprinting approaches have become popular for the recognition of conserved and potentially functional sequences. Several algorithms have been developed for the identification of conserved transcription factor binding sites (TFBSs), which are

  4. Three-dimensional binding sites volume assessment during cardiac pacing lead extraction

    Directory of Open Access Journals (Sweden)

    Bich Lien Nguyen

    2015-07-01

    Conclusions: Real-time 3D binding sites assessment is feasible and improves transvenous lead extraction outcomes. Its role as a complementary information requires extensive validation, and might be beneficial for a tailored strategy.

  5. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2017-03-17

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Binding sites for 3H-LTC4 in membranes from guinea pig ileal longitudinal muscle

    International Nuclear Information System (INIS)

    Nicosia, S.; Crowley, H.J.; Oliva, D.; Welton, A.F.

    1984-01-01

    Leutriene (LTC4) is one of the components of Slow Reacting Substance of Anaphylaxis (SRS-A) and is a potent constrictor of guinea pig ilea. The contraction is likely to be a receptor-mediated process. Here the authors report the existence of specific binding sites for 3 H-LTC4 in a crude membrane preparation from guinea pig ileal longitudinal muscle. At 4 degrees C in the presence of 20 mM Serine-borate, binding increases linearly with protein concentration, reaches equilibrium in 10 minutes, and is reversible upon addition of 3 x 10(-5) M unlabelled LTC4. The dissociation curve is consistent with the existence of more than one class of binding site. Ca++ and Mg++ greatly enhance the binding of 3 H-LTC4 at equilibrium. In the presence of 5 mM CaCl 2 and MgCl 2 not only LTC4 (IC50 10(-7)M), but also LTD4 and the SRS-A antagonist FPL 55712 can compete with 3 H-LTC4 for its binding sites. FPL 55712 only displaces 60-70% of the total amount bound, while LTC4 displaces 90-95%. These studies indicate that multiple classes of binding sites exist for 3 H-LTC4 in guinea pig ileal longitudinal muscle, and that at least part of these binding sites might be related to the ability of LTC4 to contract guinea pig ilea

  7. Radiolabelling of phoneutria nigriventer spider toxin (Tx1): a tool to study its binding site

    International Nuclear Information System (INIS)

    Santos, Raquel Gouvea dos; Diniz, Carlos Roberto; Nascimento, Marta Cordeiro; Lima, Maria Elena de

    1996-01-01

    The neurotoxin Tx1, isolated from the venom of the South American spider Phoneutria nigriventer produces tail elevation and spastic paralysis of posterior limbs after intracerebral ventricular injection in mice. Tx1 also produces ileum contraction in bioassay. We have investigated the binding of radioiodinated-Tx1 ( 125 I-Tx1) on the preparation of myenteric plexus-longitudinal muscle membrane from guinea pig ileum (MPLM) as a tool to characterize the interaction of this neurotoxin with its site. The neurotoxin Tx1 was radioiodinated with Na 125 I by the lactoperoxidase method. 125 I-Tx1 specifically binds to a single class of noninteracting binding sites of high affinity (Kd= 3.5 x 10 -10 M) and low capacity (1.2 pmol/mg protein). The specific binding increased in parallel with the protein concentration. In competition experiments the ligands of ionic channels used (sodium, potassium and calcium) did not affect the binding of 125 I-Tx1 to MPLM neither did the cholinergic ligands (hemicholinium-3, hexamethonium, d-tubocurarine and atropine). Another neurotoxin (Tx2-6, one of the isoforms of Tx2 pool) decreased toxin with MPLM and showed that toxin has a specific and saturable binding site in guinea pig ileum and this binding site appears to be related to the Tx2 site. (author)

  8. Identification of nucleic acid binding sites on translin-associated factor X (TRAX protein.

    Directory of Open Access Journals (Sweden)

    Gagan Deep Gupta

    Full Text Available Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.

  9. Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

    Science.gov (United States)

    Gupta, Gagan Deep; Kumar, Vinay

    2012-01-01

    Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

  10. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    Directory of Open Access Journals (Sweden)

    Renata Angeli

    2009-01-01

    Full Text Available A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A and Cratylia mollis (Cramoll 1 and Cramoll 1,4 did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

  11. Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro

    International Nuclear Information System (INIS)

    Leysen, J.E.

    1981-01-01

    In vitro binding studies to serotoninergic receptors were performed using 3 H-LSD, 3 H-5-HT and 3 H-spiperone. An overwiew is given on findings using these three ligands with respect to the following: localization of specific binding sites, in various animal species, the regional distribution in the brain and periphery, the subcellular and cellular distribution. Properties of the binding sites, influence of the composition of the assay medium, binding kinetic properties, receptor regulation in vivo. Identity of the binding sites, differences between site for various 3 H-ligands, pharmacological specificity of the membranous binding sites, chemical composition of the macromolecular complex constituting the binding site. Function of the receptor. Binding affinities of 44 compounds were measured in binding assays using 3 H-spiperone and 3 H-LSD with rat frontal cortex membrane preparations and using 3 H-5-HT and 3 H-LSD with rat hippocampal membrane preparations

  12. Self-Assembly of Coordinative Supramolecular Polygons with Open Binding Sites.

    Science.gov (United States)

    Zheng, Yao-Rong; Wang, Ming; Kobayashi, Shiho; Stang, Peter J

    2011-04-27

    The design and synthesis of coordinative supramolecular polygons with open binding sites is described. Coordination-driven self-assembly of 2,6-bis(pyridin-4-ylethynyl)pyridine with 60° and 120° organoplatinum acceptors results in quantitative formation of a supramolecular rhomboid and hexagon, respectively, both bearing open pyridyl binding sites. The structures were determined by multinuclear ((31)P and (1)H) NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, along with a computational study.

  13. Application of lectins to tumor imaging radiopharmaceuticals

    International Nuclear Information System (INIS)

    Kojima, Shuji; Jay, M.

    1986-01-01

    We investigated the in vitro binding of 125 I-lectins to Ehrlich ascites tumor (EAT) cells and in vivo uptake of 125 I-lectins in Ehrlich solid tumor (EST) bearing mice. In in vitro binding assays, phaseolus vulgaris agglutinin (PHA), pisum sativum agglutinin (PSA), and concanavalia agglutinin (Con A) showed a high affinity for EAT cells. The in vivo biodistribution of 125 I-lectins showed 125 I-PSA to be significantly taken up into EST tissues 24 h postinjection. After IV injection of 125 I-PSA, uptake of the radioactivity into the tumor tissues reached a maximum at 6 h, and thereafter decreased. Rapid disappearance of the radioactivity from blood and its excretion into kidney soon after injection of 125 I-PSA were observed. When compared with the biodistribution of 67 Ga-citrate in EST bearing mice 24 h postinjection, tumor to liver (T/B), tumor to muscle (T/M), and tumor to blood (T/B) ratios were superior for 125 I-PSA. At 6 h postinjection, the T/B-ratio of 125 I-PSA was 2.5, and this value may be sufficient to enable discernable diagnostic images. Our results suggest that PSA might be a useful tumor imaging radiopharmaceutical. (orig.)

  14. Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina.

    Science.gov (United States)

    Reichenbach, A; Dettmer, D; Brückner, G; Neumann, M; Birkenmeyer, G

    1985-03-22

    Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.

  15. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  16. Cellulase enzyme: Homology modeling, binding site identification and molecular docking

    Science.gov (United States)

    Selvam, K.; Senbagam, D.; Selvankumar, T.; Sudhakar, C.; Kamala-Kannan, S.; Senthilkumar, B.; Govarthanan, M.

    2017-12-01

    Cellulase is an enzyme that degrades the linear polysaccharide like cellulose into glucose by breaking the β-1,4- glycosidic bonds. These enzymes are the third largest enzymes with a great potential towards the ethanol production and play a vital role in degrading the biomass. The production of ethanol depends upon the ability of the cellulose to utilize the wide range of substrates. In this study, the 3D structure of cellulase from Acinetobacter sp. was modeled by using Modeler 9v9 and validated by Ramachandran plot. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 81.1% in the favored region, compatibility of an atomic model (3D) with amino acid sequence (1D) for the model was observed as 78.21% and 49.395% for Verify 3D and ERRAT at SAVES server. As the binding efficacy with the substrate might suggests the choice of the substrate as carbon and nitrogen sources, the cellobiose, cellotetraose, cellotetriose and laminaribiose were employed in the docking studies. The docking of cellobiose, cellotetraose, cellotetriose and laminaribiose with cellulase exhibited the binding energy of -6.1523 kJ/mol, -7.8759 kJ/mol,-6.1590 kJ/mol and -6.7185 kJ/mol, respectively. These docking studies revealed that cellulase has the greater potential towards the cellotetraose as a substrate for the high yield of ethanol.

  17. High affinity [3H]glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    International Nuclear Information System (INIS)

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D.

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea [3H] glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of [3H] glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer [3H]glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of [3H]glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats

  18. PocketMatch: A new algorithm to compare binding sites in protein structures

    Directory of Open Access Journals (Sweden)

    Chandra Nagasuma

    2008-12-01

    Full Text Available Abstract Background Recognizing similarities and deriving relationships among protein molecules is a fundamental requirement in present-day biology. Similarities can be present at various levels which can be detected through comparison of protein sequences or their structural folds. In some cases similarities obscure at these levels could be present merely in the substructures at their binding sites. Inferring functional similarities between protein molecules by comparing their binding sites is still largely exploratory and not as yet a routine protocol. One of the main reasons for this is the limitation in the choice of appropriate analytical tools that can compare binding sites with high sensitivity. To benefit from the enormous amount of structural data that is being rapidly accumulated, it is essential to have high throughput tools that enable large scale binding site comparison. Results Here we present a new algorithm PocketMatch for comparison of binding sites in a frame invariant manner. Each binding site is represented by 90 lists of sorted distances capturing shape and chemical nature of the site. The sorted arrays are then aligned using an incremental alignment method and scored to obtain PMScores for pairs of sites. A comprehensive sensitivity analysis and an extensive validation of the algorithm have been carried out. A comparison with other site matching algorithms is also presented. Perturbation studies where the geometry of a given site was retained but the residue types were changed randomly, indicated that chance similarities were virtually non-existent. Our analysis also demonstrates that shape information alone is insufficient to discriminate between diverse binding sites, unless combined with chemical nature of amino acids. Conclusion A new algorithm has been developed to compare binding sites in accurate, efficient and high-throughput manner. Though the representation used is conceptually simplistic, we demonstrate that

  19. Collectin-11/MASP complex formation triggers activation of the lectin complement pathway--the fifth lectin pathway initiation complex

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Skjoedt, Mikkel-Ole; Garred, Peter

    2013-01-01

    Collectins and ficolins are important in the clearance of endogenous and exogenous danger materials. A new human collectin-11 was recently identified in low concentration in serum in complex with mannose-binding lectin (MBL)/ficolin-associated serine proteases. Collectin-11 binds to carbohydrate...... complement complex on C. albicans. Moreover, spiking collectin-11-depleted serum, which did not mediate complement activation, with recombinant collectin-11 restored the complement activation capability. These results define collectin-11 as the fifth recognition molecule in the lectin complement pathway...

  20. [Separation of osteoclasts by lectin affinity chromatography].

    Science.gov (United States)

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  1. The binding sites for cocaine and dopamine in the dopamine transporter overlap

    DEFF Research Database (Denmark)

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L

    2008-01-01

    Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... inhibition of dopamine transport by cocaine....

  2. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    International Nuclear Information System (INIS)

    Gil, D.W.; Wolfe, B.B.

    1986-01-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [ 3 H]quinuclidinyl benzilate or [ 3 H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [ 3 H]quinuclidinyl benzilate in a biphasic manner

  3. Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

    Science.gov (United States)

    Szumańska, G; Gadamski, R

    1992-01-01

    Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

  4. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

  5. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    Science.gov (United States)

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

  6. Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux.

    Directory of Open Access Journals (Sweden)

    Soazig Le Lay

    Full Text Available Caveolin-1 (Cav1, a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs derived from wild type (WT or Cav1-deficient (Cav1(-/- animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/- cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/- MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/- cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

  7. Diversity and evolutionary relationship of nucleotide binding site ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    site-encoding disease-resistance gene analogues in sweet potato. (Ipomoea batatas Lam.) ... terminal domain of the protein, this class of R-genes can be subdivided into TIR ... from young leaflets using the modified 2.0% (w/v) cetyl trimethyl ...

  8. Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Keishi Yamasaki

    Full Text Available A wide variety of drugs bind to human serum albumin (HSA at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand and ibuprofen (site II ligand at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a 'competitive-like' allosteric interaction in the case of the defatted HSA conformer to a 'nearly independent' binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a 'competitive-like' allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to 'nearly independent' binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

  9. 2[125I]Iodomelatonin binding sites in spleens of guinea pigs

    International Nuclear Information System (INIS)

    Poon, A.M.S.; Pang, S.F.

    1992-01-01

    2-[ 125 I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8±4.12 pmol/l and binding site density (Bmax) of 0.69±0.082 fmol/mg protein at mid-light. There was no significant change in the Kd or the Bmax at mid-dark. Kinetic analysis showed a Kd of 23.13±4.81 pmol/l, in agreement to that derived from the saturation studies. The 2-[ 125 I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin much-gt N-acetylserotonin, 6-hydroxymelatonin > 5-methoxytryptamine, 5-methoxytryptophol > serotonin, 5-methoxyindole-3-acetic acid > 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan > tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction, the rest are distributed in the microsomal fraction, mitochondrial fraction and cytosolic fraction. The demonstration of 2-[ 125 I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system

  10. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  11. Copper(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization.

    Science.gov (United States)

    Ramis, Rafael; Ortega-Castro, Joaquín; Vilanova, Bartolomé; Adrover, Miquel; Frau, Juan

    2017-08-03

    The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.

  12. Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

    International Nuclear Information System (INIS)

    Kumar, K.P.; Chatterji, D.

    1990-01-01

    Terbium(III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K d between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31 P and 1 H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K d values of 4 μM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the β-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 angstrom for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the β-subunit of E. coli RNA polymerase was measured to be around 30 angstrom. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate

  13. Analysis of functional importance of binding sites in the Drosophila gap gene network model.

    Science.gov (United States)

    Kozlov, Konstantin; Gursky, Vitaly V; Kulakovskiy, Ivan V; Dymova, Arina; Samsonova, Maria

    2015-01-01

    The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. We elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

  14. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

    Science.gov (United States)

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-04-15

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

  15. Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

    Science.gov (United States)

    Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

    2008-11-21

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

  16. Antifungal activity of lectins against yeast of vaginal secretion

    Directory of Open Access Journals (Sweden)

    Bruno Severo Gomes

    2012-06-01

    Full Text Available Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256µg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

  17. Oligomycin frames a common drug-binding site in the ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

    2015-12-01

    We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

  18. The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

    Science.gov (United States)

    Horton, R W; Lowther, S; Chivers, J; Jenner, P; Marsden, C D; Testa, B

    1988-08-01

    1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites. 8. Clebopride and Delagrange 2674 are structurally dissimilar to other BDZ ligands and represent another chemical structure to probe brain BDZ binding sites.

  19. (/sup 3/H)Spiperone binding sites in brain: autoradiographic localization of multiple receptors

    Energy Technology Data Exchange (ETDEWEB)

    Palacios, J M; Niehoff, D L; Kuhar, M J [Johns Hopkins Univ., Baltimore, MD (USA). School of Medicine

    1981-01-01

    (/sup 3/H)Spiperone ((/sup 3/H)SP) binding sites were localized by light microscopic autoradiography, after in vitro labelling. The kinetic and pharmacological characteristics of these binding sites were studied in slide-mounted sections of rat forebrain, and optimal labeling conditions were defined. Autoradiograms were obtained by apposing emulsion-coated coverslips to labeled sections. Differential drug sensitivity allowed the selective displacement of (/sup 3/H)SP from dopamine receptors by ADTN, from serotonin receptors by cinanserin, from both by haloperidol and from unique spiperone sites by unlabeled spiperone. The various sites presented a differential anatomical localization. For example, only dopaminergic sites were found in the glomerular layer of the olfactory bulb; only serotonergic sites were found in lamina IV of the neocortex, and a high concentration of unique spiperone sites were found in parts of the hippocampus.

  20. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

  1. Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

    Science.gov (United States)

    Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

    2017-11-01

    We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

  2. Polymorphisms of Mannose-binding Lectin and Toll-like Receptors 2, 3, 4, 7 and 8 and the Risk of Respiratory Infections and Acute Otitis Media in Children.

    Science.gov (United States)

    Toivonen, Laura; Vuononvirta, Juho; Mertsola, Jussi; Waris, Matti; He, Qiushui; Peltola, Ville

    2017-05-01

    Mannose-binding lectin (MBL) and toll-like receptors (TLRs) are important components of the innate immune system. We assessed the susceptibility of children with genetic variants in these factors to respiratory infections, rhinovirus infections and acute otitis media. In a prospective cohort study, blood samples from 381 Finnish children were analyzed for polymorphisms in MBL2 at codons 52, 54 and 57, TLR2 Arg753Gln, TLR3 Leu412Phe, TLR4 Asp299Gly, TLR7 Gln11Leu and TLR8 Leu651Leu. Children were followed up for respiratory infections until 24 months of age with daily diaries. Polymerase chain reaction and antigen tests were used for detection of respiratory viruses from nasal swabs. Children with MBL variant genotype had a mean of 59 days with symptoms of respiratory infection per year, compared with 49 days in those with wild-type (P = 0.01). TLR8 polymorphisms were associated with an increased risk and TLR7 polymorphisms with a decreased risk of recurrent rhinovirus infections (P = 0.02 for both). TLR2 polymorphisms were associated with recurrent acute otitis media (P = 0.02). MBL polymorphisms were associated with an increased and TLR7 polymorphisms with a decreased risk of rhinovirus-associated acute otitis media (P = 0.03 and P = 0.006, respectively). Genetic polymorphisms in MBL and TLRs promote susceptibility to or protection against respiratory infections. In addition to environmental factors, genetic variations may explain why some children are more prone to respiratory infections.

  3. Lectin histochemistry of enterocytes sugar residues in the gut of the chick embryo and of the newborn.

    Science.gov (United States)

    Bryk, S Gheri; Gheri, G

    2002-01-01

    The glycoconjugates sugar residues content, distribution and changes in the enterocytes of different tracts of the developing intestine of the chick embryo and of 1-day-old chick were investigated, using a battery of seven HRP-conjugated lectins (DBA, SBA, PNA, WGA, ConA, LTA and UEAI). The results of the present research have shown the presence of a large amount of glycoconjugates sugar residues in the enterocytes of duodenal, ileal and colonic anlage, starting from the beginning of the second week of incubation. Differences were detected among the three investigated intestinal segments, as to the time of appearance of the glycoconjugates sugar residues in the enterocytes. The duodenal enterocytes showed the most precocious appearance of lectin-reactive material, followed by the ileal enterocytes and afterwards by colonic enterocytes. The duodenal enterocytes were characterised by the presence of SBA binding sites, which were not detectable in the duodenal enterocytes of the adult animal.

  4. In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

    DEFF Research Database (Denmark)

    Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme

    2017-01-01

    In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed...

  5. Carotenoid-binding sites of the major light-harvesting complex II of higher plants

    NARCIS (Netherlands)

    Croce, Roberta; Weiss, Saskia; Bassi, Roberto

    1999-01-01

    Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron

  6. Phyloscan: locating transcription-regulating binding sites in mixed aligned and unaligned sequence data.

    Science.gov (United States)

    Palumbo, Michael J; Newberg, Lee A

    2010-07-01

    The transcription of a gene from its DNA template into an mRNA molecule is the first, and most heavily regulated, step in gene expression. Especially in bacteria, regulation is typically achieved via the binding of a transcription factor (protein) or small RNA molecule to the chromosomal region upstream of a regulated gene. The protein or RNA molecule recognizes a short, approximately conserved sequence within a gene's promoter region and, by binding to it, either enhances or represses expression of the nearby gene. Since the sought-for motif (pattern) is short and accommodating to variation, computational approaches that scan for binding sites have trouble distinguishing functional sites from look-alikes. Many computational approaches are unable to find the majority of experimentally verified binding sites without also finding many false positives. Phyloscan overcomes this difficulty by exploiting two key features of functional binding sites: (i) these sites are typically more conserved evolutionarily than are non-functional DNA sequences; and (ii) these sites often occur two or more times in the promoter region of a regulated gene. The website is free and open to all users, and there is no login requirement. Address: (http://bayesweb.wadsworth.org/phyloscan/).

  7. Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea).

    Science.gov (United States)

    Ao, Jingqun; Ding, Yang; Chen, Yuanyuan; Mu, Yinnan; Chen, Xinhua

    2015-12-10

    The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1-C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca(2+) binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca(2+)-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

  8. Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea

    Directory of Open Access Journals (Sweden)

    Jingqun Ao

    2015-12-01

    Full Text Available The C-type lectin-like receptors (CTLRs play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD, an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1–C6, a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp and WYD (Trp-Tyr-Asp. Ca2+ binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus and guppy (Poecilia retitculata CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca2+-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

  9. Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules

    DEFF Research Database (Denmark)

    Thiel, S; Jensen, L; Degn, Søren Egedal

    2012-01-01

    , an important component of the innate immune system. Three proteins are produced from the MASP1 gene: MASP-1 and MASP-3 and MAp44. We present an assay specific for MASP-1, which is based on inhibition of the binding of anti-MASP-1-specific antibody to MASP-1 domains coated onto microtitre wells. MASP-1...... was found in serum in large complexes eluting in a position corresponding to ∼600 kDa after gel permeation chromatography in calcium-containing buffer and as monomers of ∼75 kDa in dissociating buffer. The concentration of MASP-1 in donor sera (n = 105) was distributed log-normally with a median value of 11...

  10. Two distinct affinity binding sites for IL-1 on human cell lines

    International Nuclear Information System (INIS)

    Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

    1989-01-01

    We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

  11. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

    Directory of Open Access Journals (Sweden)

    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  12. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun; Peng, Chengbin; Li, Yue

    2016-01-01

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  13. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  14. Mercury Binding Sites in Thiol-Functionalized Mesostructured Silica

    International Nuclear Information System (INIS)

    Billinge, Simon J.L.; McKimmey, Emily J.; Shatnawi, Mouath; Kim, HyunJeong; Petkov, Valeri; Wermeille, Didier; Pinnavaia, Thomas J.

    2005-01-01

    Thiol-functionalized mesostructured silica with anhydrous compositions of (SiO 2 ) 1-x (LSiO 1.5 ) x , where L is a mercaptopropyl group and x is the fraction of functionalized framework silicon centers, are effective trapping agents for the removal of mercuric(II) ions from water. In the present work, we investigate the mercury-binding mechanism for representative thiol-functionalized mesostructures by atomic pair distribution function (PDF) analysis of synchrotron X-ray powder diffraction data and by Raman spectroscopy. The mesostructures with wormhole framework structures and compositions corresponding to x = 0.30 and 0.50 were prepared by direct assembly methods in the presence of a structure-directing amine porogen. PDF analyses of five mercury-loaded compositions with Hg/S ratios of 0.50-1.30 provided evidence for the bridging of thiolate sulfur atoms to two metal ion centers and the formation of chain structures on the pore surfaces. We find no evidence for Hg-O bonds and can rule out oxygen coordination of the mercury at greater than the 10% level. The relative intensities of the PDF peaks corresponding to Hg-S and Hg-Hg atomic pairs indicate that the mercury centers cluster on the functionalized surfaces by virtue of thiolate bridging, regardless of the overall mercury loading. However, the Raman results indicate that the complexation of mercury centers by thiolate depends on the mercury loading. At low mercury loadings (Hg/S (le) 0.5), the dominant species is an electrically neutral complex in which mercury most likely is tetrahedrally coordinated to bridging thiolate ligands, as in Hg(SBu t ) 2 . At higher loadings (Hg/S 1.0-1.3), mercury complex cations predominate, as evidenced by the presence of charge-balancing anions (nitrate) on the surface. This cationic form of bound mercury is assigned a linear coordination to two bridging thiolate ligands.

  15. Specificity of cellular DNA-binding sites of microbial populations in a Florida reservoir

    International Nuclear Information System (INIS)

    Paul, J.H.; Pichard, S.L.

    1989-01-01

    The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of [ 3 H]- or [ 32 P]DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nuclei acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments

  16. Characterization of a second ligand binding site of the insulin receptor

    International Nuclear Information System (INIS)

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan

    2006-01-01

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

  17. Down-regulation of endothelin binding sites in rat vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Roubert, P.; Gillard, V.; Plas, P.; Chabrier, P.E.; Braquet, P.

    1990-01-01

    In cultured rat aortic smooth muscle cells, [ 125 I]endothelin (ET-1) bound to an apparent single class of high affinity recognition sites with a dissociation constant of 1.84 +/- 0.29 nmol/L and a maximum binding of 62 +/- 10.5 fmol/10(6) cells. The binding was not affected by calcium antagonists or vasoactive substances, including angiotensin II, arginine vasopressin, atrial natriuretic factor and bradykinin. Exposure of the cells to ET-1 (0.01 nmol/L to 10 nmol/L) resulted